Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol

ABSTRACT

The invention provides a non-naturally occurring microbial organism having an acetyl-CoA pathway and the capability of utilizing syngas or syngas and methanol. In one embodiment, the invention provides a non-naturally occurring microorganism, comprising one or more exogenous proteins conferring to the microorganism a pathway to convert CO, CO 2  and/or H 2  to acetyl-coenzyme A (acetyl-CoA), methyl tetrahydrofolate (methyl-THF) or other desired products, wherein the microorganism lacks the ability to convert CO or CO 2  and H 2  to acetyl-CoA or methyl-THF in the absence of the one or more exogenous proteins. For example, the microbial organism can contain at least one exogenous nucleic acid encoding an enzyme or protein in an acetyl-CoA pathway. The microbial organism is capable of utilizing synthesis gases comprising CO, CO 2  and/or H 2 , alone or in combination with methanol, to produce acetyl-CoA. The invention additionally provides a method for producing acetyl-CoA, for example, by culturing an acetyl-CoA producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme or protein in a sufficient amount to produce acetyl-CoA, under conditions and for a sufficient period of time to produce acetyl-CoA.

This application is a continuation of U.S. application Ser. No.12/358,217, filed Jan. 22, 2009, which claims the benefit of priority ofU.S. Provisional Ser. No. 61/022,804, filed Jan. 22, 2008, and U.S.Provisional Ser. No. 61/059,256, filed Jun. 5, 2008, each of which theentire contents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes andmore specifically to organisms capable of using synthesis gas or othergaseous carbon sources and methanol.

Synthesis gas (syngas) is a mixture of primarily H₂ and CO that can beobtained via gasification of any organic feedstock, such as coal, coaloil, natural gas, biomass, or waste organic matter. Numerousgasification processes have been developed, and most designs are basedon partial oxidation, where limiting oxygen avoids full combustion, oforganic materials at high temperatures (500-1500° C.) to provide syngasas a 0.5:1-3:1 H₂/CO mixture. Steam is sometimes added to increase thehydrogen content, typically with increased CO₂ production through thewater gas shift reaction.

Today, coal is the main substrate used for industrial production ofsyngas, which is traditionally used for heating and power and as afeedstock for Fischer-Tropsch synthesis of methanol and liquidhydrocarbons. Many large chemical and energy companies employ coalgasification processes on large scale and there is experience in theindustry using this technology.

In addition to coal, many types of biomass have been used for syngasproduction. Gaseous substrates such as syngas and CO₂ represent the mostinexpensive and most flexible feedstocks available for the biologicalproduction of renewable chemicals and fuels. During World War II, therewere over 1 million small scale biomass gasification units in operation,mainly in Europe, for running cars, trucks, boats, and buses. Currently,there are at least three major biomass gasification technologies thathave been or are in the process of being validated on a commercial scale(>20 million lb biomass/yr). Biomass gasification technologies are beingpracticed commercially, particularly for heat and energy generation.Integration with fuels or chemicals production is being developed andhas not yet been demonstrated widely at a commercial scale.

Overall, technology now exists for cost-effective production of syngasfrom a plethora of materials, including coal, biomass, wastes, polymers,and the like, at virtually any location in the world. The benefits ofusing syngas include flexibility, since syngas can be produced from mostorganic substances, including biomass. Another benefit is that syngas isinexpensive, costing ≦$6 per million Btu, representing raw materialcosts of ≦$0.10/lb product. In addition, there are known pathways, as inorganisms such as Clostridium spp., that utilize syngas effectively.

Despite the availability of organisms that utilize syngas, in generalthe known organisms are poorly characterized and are not well suited forcommercial development. For example, Clostridium and related bacteriaare strict anaerobes that are intolerant to high concentrations ofcertain products such as butanol, thus limiting titers andcommercialization potential. The Clostridia also produce multipleproducts, which presents separations issues in obtaining a desiredproduct. Finally development of facile genetic tools to manipulateClostridial genes is in its infancy; therefore, they are not readilyamenable to genetic engineering to improve yield or productioncharacteristics of a desired product.

Thus, there exists a need to develop microorganisms and methods of theiruse to utilize syngas or other gaseous carbon sources for the productionof desired chemicals and fuels. More specifically, there exists a needto develop microorganisms for synthesis gas utilization that also haveexisting and efficient genetic tools to enable their rapid engineeringto produce valuable products at useful rates and quantities. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving an acetyl-CoA pathway and the capability of utilizing syngas orsyngas and methanol. In one embodiment, the invention provides anon-naturally occurring microorganism, comprising one or more exogenousproteins conferring to the microorganism a pathway to convert CO, CO₂and/or H₂ to acetyl-coenzyme A (acetyl-CoA), methyl tetrahydrofolate(methyl-THF) or other desired products, wherein the microorganism lacksthe ability to convert CO or CO₂ and H₂ to acetyl-CoA or methyl-THF inthe absence of the one or more exogenous proteins. For example, themicrobial organism can contain at least one exogenous nucleic acidencoding an enzyme or protein in an acetyl-CoA pathway. The microbialorganism is capable of utilizing synthesis gases comprising CO, CO₂and/or H₂, alone or in combination with methanol, to produce acetyl-CoA.The invention additionally provides a method for producing acetyl-CoA,for example, by culturing an acetyl-CoA producing microbial organism,where the microbial organism expresses at least one exogenous nucleicacid encoding an acetyl-CoA pathway enzyme or protein in a sufficientamount to produce acetyl-CoA, under conditions and for a sufficientperiod of time to produce acetyl-CoA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary Wood-Ljungdahl pathway utilizing syngas as acarbon source. A methyl branch is depicted showing utilization of syngasto produce methyl-tetrahydrofolate (Me-THF).

FIG. 2 shows an exemplary Wood-Ljungdahl pathway using syngas as acarbon source. A carbonyl branch is depicted showing utilization ofsyngas to produce acetyl-coenzyme A (acetyl-CoA). Hydrogenase (12) isrequired to convert hydrogen from syngas into reducing equivalents thatare needed in many of the reactions depicted.

FIG. 3 shows a metabolic pathway diagram depicting the integration ofthe Wood-Ljungdahl and butanol production pathways. The transformationsthat are typically unique to organisms capable of growth on synthesisgas are: 1) CO dehydrogenase, 2) hydrogenase, 3) energy-conservinghydrogenase (ECH), and 4) bi-functional CO dehydrogenase/acetyl-CoAsynthase.

FIG. 4 shows a diagram depicting a process for utilizing syngas toproduce butanol. FIG. 4A shows a block flow diagram for a syngas tobutanol process. FIG. 4B shows details of the gasifier. ASU representsair separation unit.

FIG. 5 shows a proposed polyhydroxybutyrate (PHB) pathway modificationin R. rubrum to form 1-butanol. Bold arrows indicate reaction steps thatare introduced via heterologous expression of a 4-gene operon formingthe 1-butanol pathway from C. acetobutylicum. Abbreviations used arePHB, poly-β-hydroxybutyrate; PhbC, PHB synthase; Crt, crotonase; Bcd,butyryl-CoA dehydrogenase; Etf, electron transfer flavoprotein; AdhE2,aldehyde/alcohol dehydrogenase.

FIG. 6 shows a complete Wood-Ljungdahl pathway that allows theconversion of gases comprising CO, CO₂, and/or H₂ to acetyl-CoA, whichcan subsequently be converted to cell mass and products such as ethanolor acetate. Exemplary specific enzymatic transformations that can beengineered into a production host are numbered. Abbreviations: 10FTHF:10-formyltetrahydrofolate, 5MTHF: 5-methyltetrahydrofolate, ACTP: acetylphosphate, FOR: formate, METHF: methylene-tetrahydrofolate, MLTHF:methenyl-tetrahydrofolate, THF: tetrahydrofolate.

FIG. 7 shows a synthetic metabolic pathway that allows the conversion ofgases comprising CO, CO₂, and/or H₂ and methanol to acetyl-CoA. Thespecific enzymatic transformations that can be engineered into aproduction host are numbered. Additional abbreviation: MeOH: methanol.

FIG. 8 shows a pathway for conversion of methanol, CO and CO₂ to cellmass and fermentation products.

FIG. 9 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5)and controls of M. thermoacetica CODH (Moth_(—)1202/1203) or Mtr(Moth_(—)1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000ng).

FIG. 10 shows CO oxidation assay results. Cells (M. thermoacetica or E.coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S)were grown and extracts prepared. Assays were performed at 55° C. atvarious times on the day the extracts were prepared. Reduction ofmethylviologen was followed at 578 nm over a 120 sec time course.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to developing and using microorganismscapable of utilizing syngas or other gaseous carbon sources to produce adesired product. The invention further relates to expanding the productrange of syngas-utilizing microorganisms and generating recombinantorganisms capable of utilizing syngas to produce a desired product andoptimizing their yields, titers, and productivities. Development of arecombinant organism, for example, Escherichia coli or other organismssuitable for commercial scale up, that can efficiently utilize syngas asa substrate for growth and for chemical production providescost-advantaged processes for renewable chemical and fuel manufacturing.The organisms can be optimized and tested rapidly and at reasonablecosts.

The great potential of syngas as a feedstock resides in its ability tobe efficiently and cost-effectively converted into chemicals and fuelsof interest. Two main technologies for syngas conversion areFischer-Tropsch processes and fermentative processes. TheFischer-Tropsch (F-T) technology has been developed since World War IIand involves inorganic and metal-based catalysts that allow efficientproduction of methanol or mixed hydrocarbons as fuels. The drawbacks ofF-T processes are: 1) a lack of product selectivity, which results indifficulties separating desired products; 2) catalyst sensitivity topoisoning; 3) high energy costs due to high temperatures and pressuresrequired; and 4) the limited range of products available at commerciallycompetitive costs.

For fermentative processes, syngas has been shown to serve as a carbonand energy source for many anaerobic microorganisms that can convertthis material into products such as ethanol, acetate and hydrogen (seebelow and Table 1). The main benefits of fermentative conversion ofsyngas are the selectivity of organisms for production of singleproducts, greater tolerance to syngas impurities, lower operatingtemperatures and pressures, and potential for a large portfolio ofproducts from syngas. The main drawbacks of fermentative processes arethat organisms known to convert syngas tend to generate only a limitedrange of chemicals, such as ethanol and acetate, and are not efficientproducers of other chemicals, the organisms lack established tools forgenetic manipulation, and the organisms are sensitive to end products athigh concentrations.

The present invention relates to the generation of microorganisms thatare effective at producing desired products, including chemicals andfuels, from syngas or other gaseous carbon sources. The organisms andmethods of the present invention allow production of chemicals and fuelsat costs that are significantly advantaged over both traditionalpetroleum-based products and products derived directly from glucose,sucrose or lignocellulosic sugars. In one embodiment, the inventionprovides a non-naturally occurring microorganism capable of utilizingsyngas or other gaseous carbon sources to produce desired products inwhich the parent microorganism lacks the natural ability to utilizesyngas (see Example VIII). In such microorganisms, one or more proteinsor enzymes are expressed in the microorganism, thereby conferring apathway to utilize syngas or other gaseous carbon source to produce adesired product. In other embodiments, the invention provides anon-naturally occurring microorganism that has been geneticallymodified, for example, by expressing one or more exogenous proteins orenzymes that confer an increased efficiency of production of a desiredproduct, where the parent microorganism has the ability to utilizesyngas or other gaseous carbon source to produce a desired product.Thus, the invention relates to generating a microorganism with a newmetabolic pathway capable of utilizing syngas as well as generating amicroorganism with improved efficiency of utilizing syngas or othergaseous carbon source to produce a desired product.

The present invention additionally provides a non-naturally occurringmicroorganism expressing genes encoding enzymes that catalyze andproteins associated with the carbonyl-branch of the Wood-Ljungdahlpathway in conjunction with a MtaABC-type methyltransferase system. Suchan organism is capable of converting methanol, a relatively inexpensiveorganic feedstock that can be derived from synthesis gas, and gasescomprising CO, CO₂, and/or H₂ into acetyl-CoA, cell mass, and products.

Escherichia coli is an industrial workhorse organism with an unrivaledsuite of genetic tools. Engineering the capability to convert synthesisgas into acetyl-CoA, the central metabolite from which all cell masscomponents and many valuable products can be derived, into a foreignhost such as E. coli can be accomplished following the expression ofexogenous genes that encode various proteins of the Wood-Ljungdahlpathway. This pathway is highly active in acetogenic organisms such asMoorella thermoacetica (formerly, Clostridium thermoaceticum), which hasbeen the model organism for elucidating the Wood-Ljungdahl pathway sinceits isolation in 1942 (Fontaine et al., J Bacteriol. 43:701-715 (1942)).The Wood-Ljungdahl pathway comprises two branches: the Eastern, ormethyl, branch that allows the conversion of CO₂ tomethyltetrahydrofolate (Me-THF) and the Western, or carbonyl, branchthat allows the conversion of methyl-THF, CO, and Coenzyme-A intoacetyl-CoA (see FIGS. 1 and 2). As disclosed herein, the inventionprovides a non-naturally occurring microorganism expressing genes thatcatalyze both branches of the Wood-Ljungdahl pathway. Such an organismis capable of converting gasses comprising CO, CO₂, and/or H2 intoacetyl-CoA, cell mass, and products. The invention additionally providesa non-naturally occurring microorganism expressing genes encodingenzymes that catalyze the carbonyl-branch of the Wood-Ljungdahl pathwayin conjunction with a MtaABC-type methyltransferase system. Such anorganism is capable of converting methanol, a relatively inexpensiveorganic feedstock that can be derived from synthesis gas, and gasescomprising CO, CO₂, and/or H₂ into acetyl-CoA, cell mass, and products.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

As disclosed herein, gaseous carbon sources such as syngas comprising COand/or CO₂ can be utilized by non-naturally occurring microorganisms ofthe invention to produce a desired product. Although generallyexemplified herein as syngas, it is understood that any source ofgaseous carbon comprising CO and/or CO₂ can be utilized by thenon-naturally occurring microorganisms of the invention. Thus, theinvention relates to non-naturally occurring microorganisms that arecapable of utilizing CO and/or CO₂ as a carbon source.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a acetyl-CoA pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains one branch or the complete Wood-Ljungdahl pathway willconfer syngas utilization ability.

Thus, the non-naturally occurring microorganisms of the invention canuse syngas or other gaseous carbon sources providing CO and/or CO₂ toproduce a desired product. In the case of CO₂, additional sourcesinclude, but are not limited to, production of CO₂ as a byproduct inammonia and hydrogen plants, where methane is converted to CO₂;combustion of wood and fossil fuels; production of CO₂ as a byproduct offermentation of sugar in the brewing of beer, whisky and other alcoholicbeverages, or other fermentative processes; thermal decomposition oflimestone, CaCO₃, in the manufacture of lime, CaO; production of CO₂ asbyproduct of sodium phosphate manufacture; and directly from naturalcarbon dioxide springs, where it is produced by the action of acidifiedwater on limestone or dolomite.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within an acetyl-CoAbiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism or microorganism is intended to mean an organism thatis substantially free of at least one component as the referencedmicrobial organism is found in nature. The term includes a microbialorganism that is removed from some or all components as it is found inits natural environment. The term also includes a microbial organismthat is removed from some or all components as the microbial organism isfound in non-naturally occurring environments. Therefore, an isolatedmicrobial organism is partly or completely separated from othersubstances as it is found in nature or as it is grown, stored orsubsisted in non-naturally occurring environments. Specific examples ofisolated microbial organisms include partially pure microbes,substantially pure microbes and microbes cultured in a medium that isnon-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having acetyl-CoA biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In one embodiment, the invention provides a non-naturally occurringmicroorganism comprising one or more exogenous proteins conferring tothe microorganism a pathway to convert CO and/or CO₂ and H₂ toacetyl-coenzyme A (acetyl-CoA), wherein the microorganism lacks theability to convert CO and/or CO₂ and H₂ to acetyl-CoA in the absence ofthe one or more exogenous proteins. For example, the one or moreexogenous proteins or enzymes can be selected from cobalamidecorrinoid/iron-sulfur protein, methyltransferase, carbon monoxidedehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfidereductase and hydrogenase (see FIG. 1 and Examples VII and VIII). Themicroorganism can also express two or more, three or more, and the like,including up to all the proteins and enzymes that confer a pathway toconvert CO and/or CO₂ and H₂ to acetyl-CoA, for example, cobalamidecorrinoid/iron-sulfur protein, methyltransferase, carbon monoxidedehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfidereductase and hydrogenase.

As disclosed herein, an embodiment of the invention relates togenerating a non-naturally occurring microorganism that can utilize COand/or CO₂ as a carbon source to produce a desired product. For example,the proteins and enzymes of the carbonyl and/or methyl branch of theWood-Ljungdahl pathway (FIGS. 1 and 2) are introduced into amicroorganism that does not naturally contain the Wood-Ljungdahlenzymes. A particularly useful organism for genetically engineering aWood-Ljungdahl pathway is E. coli, which is well characterized in termsof available genetic manipulation tools as well as fermentationconditions (see Example VIII).

In another embodiment, the invention provides a non-naturally occurringmicroorganism comprising one or more exogenous proteins conferring tothe microorganism a pathway to convert synthesis gas, also known assyngas, or other gaseous carbon source, comprising CO and H₂ toacetyl-coenzyme A (acetyl-CoA), wherein the microorganism lacks theability to convert CO and H₂ to acetyl-CoA in the absence of the one ormore exogenous proteins. Such a synthesis gas or other gas can furthercomprise CO₂. Thus, a non-naturally occurring microorganism of theinvention can comprise a pathway that increases the efficiency ofconverting CO₂, CO and/or H₂ to acetyl-CoA. In addition, the inventionprovides a non-naturally occurring microorganism comprising one or moreexogenous proteins conferring to the microorganism a pathway to converta gaseous carbon source comprising CO₂ and H₂ to acetyl-CoA, wherein themicroorganism lacks the ability to convert CO₂ and H₂ to acetyl-CoA inthe absence of the one or more exogenous proteins. The gas can furthercomprise CO. As discussed herein, the exogenous proteins can be selectedfrom cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbonmonoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthasedisulfide reductase and hydrogenase.

In yet another embodiment, the invention provides a non-naturallyoccurring microorganism comprising one or more exogenous proteinsconferring to the microorganism a pathway to convert CO and/or CO₂ andH₂ to methyl-tetrahydrofolate (methyl-THF), wherein the microorganismlacks the ability to convert CO and/or CO₂ and H₂ to methyl-THF in theabsence of the one or more exogenous proteins. As disclosed herein, theone or more exogenous proteins can be selected from ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase (see FIG. 1 and Example VIII). The microorganism can alsoexpress two or more, three or more, and the like, including up to allthe proteins and enzymes that confer a pathway to convert CO and/or CO₂and H₂ to methyl-THF, including up to all of ferredoxin oxidoreductase,formate dehydrogenase, formyltetrahydrofolate synthetase,methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolatedehydrogenase and methylenetetrahydrofolate reductase.

The invention additionally provides a non-naturally occurringmicroorganism comprising one or more exogenous proteins conferring tothe microorganism a pathway to convert synthesis gas or other gaseouscarbon source comprising CO and H₂ to methyl-THF, wherein themicroorganism lacks the ability to convert CO and H₂ to methyl-THF inthe absence of the one or more exogenous proteins. The synthesis gas canfurther comprise CO₂. In addition, the invention provides anon-naturally occurring microorganism comprising one or more exogenousproteins conferring to the microorganism a pathway to convert a gaseouscarbon source comprising CO₂ and H₂ to methyl-THF, wherein themicroorganism lacks the ability to convert CO₂ and H₂ to methyl-THF inthe absence of the one or more exogenous proteins. The gaseous carbonsource can further comprise CO. As discussed above, the exogenousproteins can be selected from ferredoxin oxidoreductase, formatedehydrogenase, formyltetrahydrofolate synthetase,methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolatedehydrogenase and methylenetetrahydrofolate reductase.

Thus, the invention relates to non-naturally occurring microorganismsand methods of utilizing such microorganisms to produce a desiredproduct such as acetyl-CoA or methyl-THF from a synthesis gas or othergas comprising CO and/or CO₂ and particularly generating microorganismscapable of utilizing syngas or other gas comprising CO and/or CO₂ thatwere not previously capable of utilizing syngas or another gascomprising CO and/or CO₂ as a carbon source (see Example VIII). Further,a microorganism can be engineered to contain both the methyl andcarbonyl branches of the Wood-Ljungdahl pathway (FIGS. 1, 2 and 6). Inaddition, other desired products can also be produced by engineering themicroorganisms to produce a desired product by expressing proteins orenzymes capable of producing a desired product, for example, producing aproduct having acetyl-CoA or methyl-THF as a precursor (see FIG. 3). Asdisclosed herein, such microorganisms can be generated by expressingproteins or genes that confer a desired metabolic pathway or bydetermining deletions that can drive metabolism towards a desiredproduct.

In addition, the invention provides a non-naturally occurringmicroorganism comprising a genetic modification conferring to themicroorganism an increased efficiency of producing acetyl-CoA from COand/or CO₂ and H₂ relative to the microorganism in the absence of thegenetic modification, wherein the microorganism comprises a pathway toconvert CO and/or CO₂ and H₂ to acetyl-CoA. In such a microorganism, thegenetic modification can comprise expression of one or more nucleic acidmolecules encoding one or more exogenous proteins, whereby expression ofthe one or more exogenous proteins increases the efficiency of producingacetyl-CoA from CO and/or CO₂ and H₂. The one or more exogenous proteinscan be selected from cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase, including up toall such proteins, as disclosed herein. Such a non-naturally occurringmicroorganism can alternatively or additionally have a geneticmodification comprising one or more gene disruptions, whereby the one ormore gene disruptions increases the efficiency of producing acetyl-CoAfrom CO and/or CO₂ and H₂. In addition, the invention provides anon-naturally occurring microorganism comprising a genetic modificationconferring an increased efficiency of producing methyl-THF or otherdesired products using the methods disclosed herein. Thus, the inventionadditionally relates to improving the efficiency of production of adesired product in a microorganism already having the ability to producethe desired product from syngas or other gases comprising CO and/or CO₂.

The invention also relates to a non-naturally occurring microorganismcomprising one or more proteins conferring utilization of syngas orother gas comprising CO and/or CO₂ as a carbon source to themicroorganism, wherein the microorganism lacks the ability to utilizethe carbon source in the absence of the one or more proteins conferringutilization of CO and/or CO₂. Further, the invention provides anon-naturally occurring microorganism comprising one or more proteinsconferring utilization of carbon monoxide and/or carbon dioxide as acarbon source to the microorganism, wherein the microorganism lacks theability to utilize the carbon source in the absence of the one or moreproteins. In yet another embodiment, the invention provides anon-naturally occurring microorganism, comprising one or more proteinsconferring utilization of CO and/or CO₂, in combination with H₂, as acarbon source to the microorganism, wherein the microorganism lacks theability to utilize the carbon source in the absence of the one or moreproteins. The invention additionally provides a non-naturally occurringmicroorganism comprising one or more proteins conferring utilization ofCO, in combination with H₂ and CO₂, as a carbon source to themicroorganism, wherein the microorganism lacks the ability to utilizethe carbon source in the absence of the one or more proteins. Such amicroorganism can be used to produce a desired product from the carbonsource, for example, methyl-tetrahydrofolate or acetyl-coenzyme A(acetyl-CoA) or other desired products, as disclosed herein, includingproducts synthesized from acetyl-CoA or methyl-THF. Such a non-naturallyoccurring microorganism can express one or more exogenous proteins thatincrease production of the product, as disclosed herein (see FIGS. 1 and2).

The invention further provides a non-naturally occurring microorganismcomprising one or more exogenous proteins conferring utilization ofsyngas or other gaseous carbon source to the microorganism, wherein themicroorganism has the ability to utilize the carbon source in theabsence of the one or more exogenous proteins, whereby expression of theone or more exogenous proteins increases the efficiency of utilizationof the carbon source. Additionally the invention provides anon-naturally occurring microorganism comprising one or more exogenousproteins conferring utilization of carbon monoxide as a carbon source tothe microorganism, wherein the microorganism has the ability to utilizethe carbon source in the absence of the one or more exogenous proteins,whereby expression of the one or more exogenous proteins increases theefficiency of utilization of the carbon source.

In yet another embodiment, the invention provides a non-naturallyoccurring microorganism comprising one or more exogenous proteinsconferring utilization of CO and/or CO₂, in combination with H₂, as acarbon source to the microorganism, wherein the microorganism has theability to utilize the carbon source in the absence of the one or moreexogenous proteins, whereby expression of the one or more exogenousproteins increases the efficiency of utilization of the carbon source.Additionally provided is a non-naturally occurring microorganismcomprising one or more exogenous proteins conferring utilization of CO,in combination with H₂ and CO₂, as a carbon source to the microorganism,wherein the microorganism has the ability to utilize the carbon sourcein the absence of the one or more exogenous proteins, whereby expressionof the one or more exogenous proteins increases the efficiency ofutilization of the carbon source. Such a microorganism can be used toproduce a desired product such as acetyl-CoA, methyl-THF or otherdesired products from the carbon source, as disclosed herein.

The invention also provides a non-naturally occurring microbial organismcapable of producing acetyl-CoA utilizing methanol and syngas. Thus, themicrobial organism is capable of utilizing methanol and CO, CO₂ and/orH₂, for example, CO₂, CO₂ and H₂, CO, CO and H₂, CO₂ and CO, or CO₂, COand H₂, to produce acetyl-CoA. Since acetyl-CoA is produced in mostmicrobial organisms, it is understood that a non-naturally occurringmicrobial organism of the invention that is capable of producingacetyl-CoA is one that has been engineered to include a desired pathway.Furthermore, the microbial organism is engineered to utilize methanoland syngas to produce acetyl-CoA (see Examples). In one embodiment, theinvention provides a non-naturally occurring microbial organism havingan acetyl-coenzyme A (acetyl-CoA) pathway comprising at least oneexogenous nucleic acid encoding an acetyl-CoA pathway enzyme or proteinexpressed in a sufficient amount to produce acetyl-CoA, the acetyl-CoApathway comprising methanol-methyltransferase and acetyl-CoAsynthase/carbon monoxide dehydrogenase. In such a non-naturallyoccurring microbial organism, the acetyl-CoA pathway can confer theability to convert CO₂, CO and/or H₂, that is, a combination thereof, toacetyl Co-A. The methanol-methyltransferase activity of such anacetyl-CoA pathway can comprise, for example, an enzyme or proteinselected from methanol methyltransferase, corrinoid protein (such asMtaC) and methyltetrahydrofolate:corrinoid protein methyltransferase(such as MtaA) (see Examples II and III). The acetyl-CoA synthase/carbonmonoxide dehydrogenase activity of such an acetyl-CoA pathway cancomprise, for example, an enzyme or protein selected frommethyltetrahydrofolate:corrinoid protein methyltransferase (such asAcsE), corrinoid iron-sulfur protein (such as AcsD), nickel-proteinassembly protein (such as AcsF), ferredoxin, acetyl-CoA synthase, carbonmonoxide dehydrogenase and nickel-protein assembly protein (such asCooC) (see Examples II and III). As disclosed herein, two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, and so forth, nucleic acids encoding anacetyl-CoA pathway can be expressed in a non-naturally occurringmicrobial organism of the invention. In a particular embodiment, thenon-naturally occurring microbial organism can comprise ten exogenousnucleic acids that encode a methanol-methyltransferase comprisingmethanol methyltransferase, corrinoid protein (such as MtaC) andmethyltetrahydrofolate:corrinoid protein methyltransferase (such asMtaA) and an acetyl-CoA synthase/carbon monoxide dehydrogenasecomprising methyltetrahydrofolate:corrinoid protein methyltransferase(such as AcsE), corrinoid iron-sulfur protein (such as AcsD),nickel-protein assembly protein (such as CooC), ferredoxin, acetyl-CoAsynthase, carbon monoxide dehydrogenase and nickel-protein assemblyprotein (such as AcsF).

In yet another embodiment, the non-naturally occurring microbialorganism can further comprise pyruvate ferredoxin oxidoreductase. Forexample, the pyruvate ferredoxin oxidoreductase can be encoded by anexogenous nucleic acid. In still another embodiment, the non-naturallyoccurring microbial organism can further comprise hydrogenase, which canbe encoded by an endogenous or exogenous nucleic acid, as disclosedherein (see Examples II and III).

As disclosed herein, a non-naturally occurring microbial organism cancontain, for example, at least one exogenous nucleic acid that is aheterologous nucleic acid. As further disclosed herein, thenon-naturally occurring microbial organism can be grown, for example, ina substantially anaerobic culture medium.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more acetyl-CoAbiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particularacetyl-CoA biosynthetic pathway can be expressed. For example, if achosen host is deficient in one or more enzymes or proteins for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve acetyl-CoA biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as acetyl-CoA.

Depending on the acetyl-CoA biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed acetyl-CoA pathway-encoding nucleic acid and up to allencoding nucleic acids for one or more acetyl-CoA biosynthetic pathways.For example, acetyl-CoA biosynthesis can be established in a hostdeficient in a pathway enzyme or protein through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes or proteins of a acetyl-CoA pathway, exogenous expression of allenzyme or proteins in the pathway can be included, although it isunderstood that all enzymes or proteins of a pathway can be expressedeven if the host contains at least one of the pathway enzymes orproteins. For example, exogenous expression of all enzymes or proteinsin a pathway for production of acetyl-CoA can be included, such as themethanol-methyltransferase, which can include methanolmethyltransferase, corrinoid protein (such as MtaC) andmethyltetrahydrofolate:corrinoid protein methyltransferase (such asMtaA), and the acetyl-CoA synthase/carbon monoxide dehydrogenase, whichcan include methyltetrahydrofolate:corrinoid protein methyltransferase(such as AcsE), corrinoid iron-sulfur protein (such as AcsD),nickel-protein assembly protein (such as AcsF), ferredoxin, acetyl-CoAsynthase, carbon monoxide dehydrogenase and nickel-protein assemblyprotein (such as CooC).

In another embodiment, in a pathway for producing acetyl-CoA from syngasor other gaseous carbon source, one or more proteins in the biosyntheticpathway can be selected from cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase (see FIG. 2 andExamples VII and VIII). In a pathway for producing methyl-THF, one ormore proteins in the biosynthetic pathway can be selected fromferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase (see FIG. 1 and Example VIII). In addition, genes that encodethe enzymes required to produce both acetyl-CoA and methyl-THF can beintroduced into a microorganism (see FIG. 3 and Example VIII). Metabolicpathways for production of additional desired products, includingsuccinate, 4-hydroxybutyrate and 1,4-butanediol are described, forexample, in U.S. application Ser. No. 11/891,602, filed Aug. 10, 2007,and WO/2008/115840 (see Example VIII).

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the acetyl-CoApathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, four, five, six, seven, eight, nine or up to allnucleic acids encoding the enzymes or proteins constituting a acetyl-CoAbiosynthetic pathway disclosed herein. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize acetyl-CoAbiosynthesis or that confer other useful functions onto the hostmicrobial organism. One such other functionality can include, forexample, augmentation of the synthesis of one or more of the acetyl-CoApathway precursors such as methanol.

Generally, a host microbial organism is selected such that it producesthe precursor of an acetyl-CoA pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. A host organism canbe engineered to increase production of a precursor, as disclosedherein. In addition, a microbial organism that has been engineered toproduce a desired precursor can be used as a host organism and furtherengineered to express enzymes or proteins of an acetyl-CoA pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize acetyl-CoA. In this specific embodiment it canbe useful to increase the synthesis or accumulation of an acetyl-CoApathway product to, for example, drive acetyl-CoA pathway reactionstoward acetyl-CoA production. Increased synthesis or accumulation can beaccomplished by, for example, overexpression of nucleic acids encodingone or more of the above-described acetyl-CoA pathway enzymes orproteins. Over expression the enzyme or enzymes and/or protein orproteins of the acetyl-CoA pathway can occur, for example, throughexogenous expression of the endogenous gene or genes, or throughexogenous expression of the heterologous gene or genes. Therefore,naturally occurring organisms can be readily generated to benon-naturally occurring microbial organisms of the invention, forexample, producing acetyl-CoA, through overexpression of one, two,three, four, five, six, seven, eight, nine, or ten, that is, up to allnucleic acids encoding acetyl-CoA biosynthetic pathway enzymes orproteins. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the acetyl-CoA biosyntheticpathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, an acetyl-CoA biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer acetyl-CoAbiosynthetic capability. For example, a non-naturally occurringmicrobial organism having a acetyl-CoA biosynthetic pathway can compriseat least two exogenous nucleic acids encoding desired enzymes orproteins, such as the combination of methanol methyltransferase andcorrinoid protein; methanol methyltransferase andmethyltetrahydrofolate:corrinoid protein methyltransferase; corrinoidprotein and corrinoid iron-sulfur protein; nickel-protein assemblyprotein and ferredoxin, and the like. Thus, it is understood that anycombination of two or more enzymes or proteins of a biosynthetic pathwaycan be included in a non-naturally occurring microbial organism of theinvention. Similarly, it is understood that any combination of three ormore enzymes or proteins of a biosynthetic pathway can be included in anon-naturally occurring microbial organism of the invention, forexample, methanol methyltransferase, corrinoid iron-sulfur protein (suchas AcsD) and acetyl-CoA synthase; corrinoid protein (such as MtaC),carbon monoxide dehydrogenase and nickel-protein assembly protein (suchas CooC or AcsF); methyltetrahydrofolate:corrinoid proteinmethyltransferase (such as AcsE), ferredoxin and acetyl-CoA synthase,and so forth, as desired, so long as the combination of enzymes and/orproteins of the desired biosynthetic pathway results in production ofthe corresponding desired product. Similarly, any combination of four,five, six, seven, eight, nine or more enzymes or proteins of abiosynthetic pathway as disclosed herein can be included in anon-naturally occurring microbial organism of the invention, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct.

In addition to the biosynthesis of acetyl-CoA as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce acetyl-CoA other than use of the acetyl-CoA producers is throughaddition of another microbial organism capable of converting anacetyl-CoA pathway intermediate to acetyl-CoA. One such procedureincludes, for example, the fermentation of a microbial organism thatproduces an acetyl-CoA pathway intermediate. The acetyl-CoA pathwayintermediate can then be used as a substrate for a second microbialorganism that converts the acetyl-CoA pathway intermediate toacetyl-CoA. The acetyl-CoA pathway intermediate can be added directly toanother culture of the second organism or the original culture of theacetyl-CoA pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, acetyl-CoA. Inthese embodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis ofacetyl-CoA can be accomplished by constructing a microbial organism thatcontains biosynthetic pathways for conversion of one pathwayintermediate to another pathway intermediate or the product.Alternatively, acetyl-CoA also can be biosynthetically produced frommicrobial organisms through co-culture or co-fermentation using twoorganisms in the same vessel, where the first microbial organismproduces an acetyl-CoA intermediate and the second microbial organismconverts the intermediate to acetyl-CoA.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce acetyl-CoA. In addition,since acetyl-CoA is a precursor of other desirable products, anon-naturally occurring microbial organism of the invention can be usedas a host organism into which other desired pathways utilizingacetyl-CoA as a precursor or intermediate can be conferred, as desired.

Sources of encoding nucleic acids for an acetyl-CoA pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Escherichiacoli, Methanosarcina barkeri, Methanosarcina acetivorans, Moorellathermoacetica, Carboxydothermus hydrogenoformans, Rhodospirillum rubrum,Acetobacterium woodii, Butyribacterium methylotrophicum, Clostridiumautoethanogenum, Clostridium carboxidivorans, Clostridium ljungdahlii,Eubacterium limosum, Oxobacter pfennigii, Peptostreptococcus productus,Rhodopseudomonas palustris P4, Rubrivivax gelatinosus, Citrobacter spY19, Methanosarcina acetivorans C2A, Methanosarcina barkeri,Desulfosporosinus orientis, Desulfovibrio desulfuricans, Desulfovibriovulgaris, Moorella thermoautotrophica, Carboxydibrachium pacificus,Carboxydocella thermoautotrophica, Thermincola carboxydiphila,Thermolithobacter carboxydivorans, Thermosinus carboxydivorans,Methanothermobacter thermoautotrophicus, Desulfotomaculumcarboxydivorans, Desulfotomaculum kuznetsovii, Desulfotomaculumnigrificans, Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum,Syntrophobacter fumaroxidans, Clostridium acidurici, Desulfovibrioafricanus, and the like, as well as other exemplary species disclosedherein or available as source organisms for corresponding genes.However, with the complete genome sequence available for now more than550 species (with more than half of these available on public databasessuch as the NCBI), including 395 microorganism genomes and a variety ofyeast, fungi, plant, and mammalian genomes, the identification of genesencoding the requisite acetyl-CoA biosynthetic activity for one or moregenes in related or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations enabling biosynthesis of acetyl-CoA described herein withreference to a particular organism such as E. coli can be readilyapplied to other microorganisms, including prokaryotic and eukaryoticorganisms alike. Given the teachings and guidance provided herein, thoseskilled in the art will know that a metabolic alteration exemplified inone organism can be applied equally to other organisms.

In some instances, such as when an alternative acetyl-CoA biosyntheticpathway exists in an unrelated species, acetyl-CoA biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizeacetyl-CoA.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger andPichia pastoris. E. coli is a particularly useful host organisms sinceit is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae. Exemplary acetogens suitable as hostorganisms include, but are not limited to, Rhodospirillum rubrum,Moorella thermoacetica and Desulfitobacterium hafniense (see Examples).

Methods for constructing and testing the expression levels of anon-naturally occurring acetyl-CoA-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofacetyl-CoA can be introduced stably or transiently into a host cellusing techniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore acetyl-CoA biosynthetic pathway encoding nucleic acids asexemplified herein operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the microbial host organisms of the invention include, for example,plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration into a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

The invention additionally provides a method for producing acetyl-CoA byculturing a non-naturally occurring microbial organism of the inventionhaving an acetyl-CoA pathway. The acetyl-CoA pathway can comprise, forexample, at least one exogenous nucleic acid encoding an acetyl-CoApathway enzyme or protein expressed in a sufficient amount to produceacetyl-CoA, under conditions and for a sufficient period of time toproduce acetyl-CoA, the acetyl-CoA pathway comprisingmethanol-methyltransferase and acetyl-CoA synthase/carbon monoxidedehydrogenase. In such an acetyl-CoA pathway, themethanol-methyltransferase can comprise an enzyme or protein selectedfrom methanol methyltransferase, corrinoid protein (such as MtaC) andmethyltetrahydrofolate:corrinoid protein methyltransferase (MtaA).Further, in such an acetyl-CoA pathway, the acetyl-CoA synthase/carbonmonoxide dehydrogenase can comprise an enzyme or protein selected frommethyltetrahydrofolate:corrinoid protein methyltransferase (such asAcsE), corrinoid iron-sulfur protein (such as AcsD), nickel-proteinassembly protein (such as AcsF), ferredoxin, acetyl-CoA synthase, carbonmonoxide dehydrogenase and nickel-protein assembly protein (such asCooC). A non-naturally occurring microbial organism can be in asubstantially anaerobic culture medium. In a particular embodiment, thenon-naturally occurring microbial organism can be cultured in thepresence of CO2, CO and/or H2, that is, a combination thereof, andmethanol. The non-naturally occurring microbial organism can furthercomprise pyruvate ferredoxin oxidoreductase, which can be expressed byan exogenous nucleic acid. The non-naturally occurring microbialorganism can also further comprise hydrogenase, for example, encoded byan endogenous or exogenous nucleic acid.

In another embodiment, the non-naturally occurring microbial organismcan be cultured in the presence of an electron acceptor, for example,nitrate, in particular under substantially anaerobic conditions (seeExample III). It is understood that an appropriate amount of nitrate canbe added to a microbial culture to achieve a desired increase inbiomass, for example, 1 mM to 100 mM nitrate, or lower or higherconcentrations, as desired, so long as the amount added provides asufficient amount of electron acceptor for the desired increase inbiomass. Such amounts include, but are not limited to, 5 mM, 10 mM, 15mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, as appropriate to achieve adesired increase in biomass.

Suitable purification and/or assays to test for the production ofacetyl-CoA can be performed using well known methods. Suitablereplicates such as triplicate cultures can be grown for each engineeredstrain to be tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art (seeExample III).

The acetyl-CoA, or products derived from acetyl-CoA, can be separatedfrom other components in the culture using a variety of methods wellknown in the art. Products derived from acetyl-CoA include, but are notlimited to, ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol,succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid,3-hydroxypropionic acid, lactic acid, methacrylic acid, adipic acid, andacrylic acid. Such separation methods include, for example, extractionprocedures as well as methods that include continuous liquid-liquidextraction, pervaporation, membrane filtration, membrane separation,reverse osmosis, electrodialysis, distillation, crystallization,centrifugation, extractive filtration, ion exchange chromatography, sizeexclusion chromatography, adsorption chromatography, andultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the acetyl-CoA producers can be cultured forthe biosynthetic production of acetyl-CoA, or products derived fromacetyl-CoA.

For the production of acetyl-CoA, the recombinant strains are culturedin a medium with a carbon and energy source of methanol and gasescomprising CO, CO₂ and/or H₂ and other essential nutrients. It is highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in U.S. patent application Ser. No. 11/891,602, filed Aug.10, 2007, and WO/2008/115840. Fermentations can be performed in a batch,fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose and starch.Other sources of carbohydrate include, for example, renewable feedstocksand biomass. Exemplary types of biomasses that can be used as feedstocksin the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of acetyl-CoA.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that expresses intracellular orsecretes the biosynthesized compounds of the invention when grown on acarbon source such as a carbohydrate, methanol, and gases comprising CO,CO₂, and/or H₂. Such compounds include, for example, acetyl-CoA and anyof the intermediate metabolites in the acetyl-CoA pathway, and productsderived from acetyl-CoA including ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, methacrylicacid, adipic acid, and acrylic acid. All that is required is to engineerin one or more of the required enzyme or protein activities to achievebiosynthesis of the desired compound or intermediate including, forexample, inclusion of some or all of the acetyl-CoA biosyntheticpathways. Accordingly, the invention provides a non-naturally occurringmicrobial organism that produces acetyl-CoA when grown on a carbohydrateor other carbon source and produces and/or secretes any of theintermediate metabolites shown in the acetyl-CoA pathway or producesand/or secretes a product derived from acetyl-CoA when grown on acarbohydrate or other carbon source. The acetyl-CoA producing microbialorganisms of the invention can initiate synthesis from an intermediate,for example, 5-methyl-tetrahydrofolate (Me-THF).

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding an acetyl-CoApathway enzyme or protein in sufficient amounts to produce acetyl-CoA.It is understood that the microbial organisms of the invention arecultured under conditions sufficient to produce acetyl-CoA. Followingthe teachings and guidance provided herein, the non-naturally occurringmicrobial organisms of the invention can achieve biosynthesis ofacetyl-CoA resulting in intracellular concentrations between about0.001-200 mM or more. Generally, the intracellular concentration ofacetyl-CoA is between about 3-150 mM, particularly between about 5-125mM and more particularly between about 8-100 mM, including about 10 mM,20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between andabove each of these exemplary ranges also can be achieved from thenon-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007, andWO/2008/115840. Any of these conditions can be employed with thenon-naturally occurring microbial organisms as well as other anaerobicconditions well known in the art. Under such anaerobic conditions, theacetyl-CoA producers can synthesize acetyl-CoA at intracellularconcentrations of 5-10 mM or more as well as all other concentrationsexemplified herein. It is understood that the above description refersto intracellular concentrations, and acetyl-CoA producing microbialorganisms can produce acetyl-CoA intracellularly. In addition, a productderived from acetyl-CoA can be produced intracellularly and/or secreted.Such products include, but are not limited to, ethanol, butanol,isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid,malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid,methacrylic acid, adipic acid, and acrylic acid

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of acetyl-CoA includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refers to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of acetyl-CoA. Exemplary growthprocedures include, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of acetyl-CoA. Generally, and as with non-continuous cultureprocedures, the continuous and/or near-continuous production ofacetyl-CoA will include culturing a non-naturally occurring acetyl-CoAproducing organism of the invention in sufficient nutrients and mediumto sustain and/or nearly sustain growth in an exponential phase.Continuous culture under such conditions can be include, for example, 1day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culturecan include 1 week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of acetyl-CoA can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the acetyl-CoAproducers of the invention for continuous production of substantialquantities of acetyl-CoA, the acetyl-CoA producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemicalconversion to convert the product to other compounds, if desired.

At least thirty different wild-type organisms have been isolated throughthe years and shown to grow on syngas or components of syngas, includingmicroorganisms capable of converting syngas to ethanol (Vega et al.,Appl. Biochem. Biotechnol. 20/21:781-797 (1989)) (see Table 1).Candidate organisms for improved syngas fermentation include acetogens,phototrophs, sulfate reducing bacteria, and methanogens, which canutilize CO and/or CO₂/H₂ as the sole carbon and energy source (Sipma etal., Crit. Rev. Biotechnol. 26:41-65. (2006)). The mesophilic acetogenClostridium carboxidivorans represents one of the most promisingorganisms for a syngas-to-chemicals platform as it has fast doublingtimes and have been shown to naturally produce ethanol and smallquantities of butanol during growth on syngas (Henstra et al., Curr.Opin. Biotechnol. 18:200-206 (2007)). Genetic tools can be developed forthis organism. The hydrogenic purple nonsulfur bacteria, Rhodospirillumrubrum, for which genetic tools exist for targeted gene deletions orinsertions, is another good candidate organism for development of syngasutilization to produce desired products, although it naturally produceshydrogen from syngas and so metabolic changes can be engineered, asrequired.

The metabolism of some syngas utilizing organisms is known. For example,acetogens such as C. carboxidivorans can grow in the presence of CO orCO₂ by utilizing the Wood-Ljungdahl pathway, even in the absence ofglucose, as long as hydrogen is present to supply the necessary reducingequivalents. The Wood-Ljungdahl pathway is illustrated in FIG. 3 (seealso FIGS. 1 and 2) and shows the capacity of acetogens to utilize CO asthe sole carbon and energy source through the production of the keymetabolic intermediate acetyl-CoA. Specifically, CO can be oxidized toproduce reducing equivalents and CO₂, or can be directly assimilatedinto acetyl-CoA, which is subsequently converted to either biomass ormetabolites. Importantly, acetyl-CoA is a key metabolic intermediatethat can serve as a precursor to a wide range of metabolites and otherchemical entities. Hence, the ability of a microorganism to produceacetyl-CoA from syngas or other gaseous carbon source allows engineeringof syngas-utilizing organisms, or organisms capable of utilizing othergaseous carbon sources, for production of a wide range of chemicals andfuels as desired products.

In order to characterize the use of syngas or other gaseous carbonsources as a viable feedstock for the commercial production of chemicalsand fuels through fermentation, feasibility studies are performed toaddress key questions and challenges associated with current systems.Preliminary metabolic modeling efforts have indicated that conversion ofsyngas to chemicals can be thermodynamically very favorable, and thatspecific chemicals can be made as the exclusive product. Not only doesthis reduce downstream processing needs, but also maximizes productyield. Furthermore, production of a desired product can begrowth-associated, so that the fermentation can be done continuously, ifdesired. Because continuous processes are maintained at high cellconcentration, and avoid batch turnaround time, they are moreeconomically favorable.

As disclosed herein, the present invention relates to the development ofmicroorganisms capable of utilizing syngas or other gaseous carbonsources, allowing the efficient conversion of CO and/or CO₂ to chemicalproducts in high yield, titer, and productivity. One exemplary usefulcommercial embodiment relates to the development of an organism that canachieve production of a specific chemical with yields ≧80% oftheoretical maximum, product tolerance ≧50 g/L, titers ≧50 g/L andproductivity of at least 2 g/L/h. Although these criteria areparticularly useful commercially, it is understood that an organismcapable of achieving less than any or all of these criteria is alsouseful in the invention. For example, an organism can achieve productionof a specific chemical with yields greater than or equal to any of 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, and so forth solong as sufficient yields are achieved for a desired application.Similarly, an organism can achieve product tolerance greater than orequal to any of 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 15 g/L,10 g/L, and so forth so long as sufficient yields are achieved for adesired application. Moreover, an organism can achieve titers greaterthan or equal to any of 200 g/L, 190 g/L, 180 g/L, 170 g/L, 160 g/L, 150g/L, 140 g/L, 130 g/L, 120 g/L, 110 g/L, 100 g/L, 90 g/L, 80 g/L, 70g/L, 60 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 15g/L, 10 g/L, and so forth so long as sufficient yields are achieved fora desired application. In addition, an organism can achieve productivityof at least any of 1.5 g/L/h, 1 g/L/h, 0.5 g/L/h, and so forth so longas sufficient yields are achieved for a desired application.

As disclosed herein, the hypothetical analysis of butanol as a productfrom syngas utilization indicates that the ability to efficientlyutilize cheap and readily available syngas as a feedstock and can leadto processes that potentially are ≧50% cost-advantaged over currentpetrochemical processes, especially in view of the low cost of syngas asa feedstock. In addition to low cost, syngas is an abundant and flexiblesubstrate that can be produced from coal and many types of biomass,including energy crops such as switchgrass, as well as waste productssuch as wood waste, agricultural waste, dairy waste, and municipal solidwaste. Thus, the ability to generate organisms capable of utilizingsyngas or other gaseous carbon sources to produce a desired productallows production from almost any biomass source. This feature obviatesthe need to develop different processes specific for each type ofbiomass used for biofuel or chemical production. The use of wasteproducts for the production of syngas can also be utilized to decreaseenvironmental pollutants and alleviate serious disposal problems ofbiowaste materials. In addition, syngas as a feedstock does not sufferfrom a feed versus fuel controversy associated with, for example,corn-based ethanol production. Given the broad range of substratesavailable for syngas production, the supply and cost structure of thisfeedstock is expected to remain relatively stable from year to year.Finally, syngas is used extensively for heating and energy and cantherefore be used as a source of biomass-derived energy that cansupplement or eliminate the need for petroleum-based energy forproduction, providing additional cost savings.

Although exemplified in various embodiments herein with butanol as adesired product, it is understood that any product capable of beingproduced by a microorganism of the invention can be generated andutilized to produce the product, as desired. Generally, desired productsinclude but are not limited to hydrocarbons useful in chemical synthesisor as a fuel. Exemplary desired products include but are not limited tomethanol, ethanol, butanol, acetate, butyrate, lactate, succinate,4-hydroxybutyrate, 1,4-butanediol, and the like.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of acetyl-CoA or productsderived from acetyl-CoA.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007, andWO/2008/115840.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Organisms and Pathways for Syngas Fermentation

This example describes organisms capable of utilizing syngas andexemplary pathways.

At least thirty different organisms have been isolated through the yearsand shown to grow on syngas or components of syngas such as CO, CO₂, andH₂ (Henstra et al., Curr. Opin. Biotechnol. 18:200-206 (2007); Sipma etal., Crit. Rev. Biotechnol. 26:41-65 (2006)). Table 1 provides examplesof such organisms as well as a number of their properties such as theiroptimal temperature for growth, optimal pH for growth, doubling time,product profile, and physiological group.

TABLE 1 Examples of CO utilizing species and their physiologicalcharacteristics. Physiological T Species Characterization (° C.) pHt_(d)(h) Products Mesophillic Bacteria Acetobacterium woodii Acetogenic30 6.8 13 Acetate Butyribacterium Acetogenic 37 6 12-20 Acetate,ethanol, metholytrophicum butyrate, butanol Clostridium Acetogenic 375.8-6.0 nr Acetate, ethanol autoethanogenum Clostridium Aceteogenic 386.2 6.25 Acetate, ethanol, carboxidivorans butyrate, butanol Clostridiumljungdahlii Acetogenic 37 6 3.8 Acetate, ethanol Eubacterium limosumAcetogenic 38-39 7.0-7.2 7 Acetate Oxobacter pfennigii Acetogenic 36-387.3 13.9 Acetate, n-butyrate Peptostrepococcus Acetogenic 37 7 1.5Acetate productus Rhodopseudomonas Hydrogenogenic, 30 nr 23 H₂ palustrisP4 Phototroph Rhodospirillum rubrum Hydrogenogenic, 30 6.8 8.4 H₂Phototroph Rubrivivax gelatinosus Hydrogenogenic, 34 6.7-6.9 6.7 H₂Phototroph Citrobacter sp Y19 Hydrogenogenic, 30-40 5.5-7.5 8.3 H₂Facultative Anaerobe Methanosarcina Methanogenic 37 7 24 Acetate,formate, acetivorans C2A CH₄ Methanosarcina barkeri Methanogenic 37 7.465 CH₄, CO₂ Desulfosporosinus Sulfate reducing 35 7 nr H₂S, CO₂ orientisbacteria Desulfovibrio Sulfate reducing 37 nr nr H₂, CO₂, H₂Sdesulfuricans bacteria Desulfovibrio vulgaris Sulfate reducing 37 nr nrH₂, CO₂, H₂S bacteria Thermophillic Bacteria Moorella thermoaceticaAcetogenic 55 6.5-6.8 10 Acetate Moorella Acetogenic 58 6.1 7 Acetatethermoautotrophica Carboxydibrachium Hydrogenogenic, 70 6.8-7.1 7.1 H₂pacificus Obligate Anearobe Carboxydocella Hydrogenogenic, 58 7 1.1 H₂thermoautotrophica Obligate Anearobe Carboxydothermus Hydrogenogenic,70-72 6.8-7.0 2 H₂ hydrogenoformans Obligate Anearobe ThermincolaHydrogenogenic, 55 8 1.3 H₂ carboxydiphila Obligate AnearobeThermolithobacter Hydrogenogenic, 70 7 8.3 H₂ carboxydivorans ObligateAnearobe Thermosinus Hydrogenogenic, 60 6.8-7.0 1.2 H₂ carboxydivoransObligate Anearobe Methanothermobacter Methanogenic 65 7.4 140 CH₄,thermoautotrophicus CO₂ Desulfotomaculum Sulfate reducing 55 7 1.7 H₂,H₂S carboxydivorans bacteria Desulfotomaculum Sulfate reducing 60 7 nrAcetate, kuznetsovil bacteria H₂S Desulfotomaculum Sulfate reducing 55 7nr H₂S, nigrificans bacteria CO₂ Desulfotomaculum Sulfate reducing 55 7nr Acetate, thermobenzoicum bacteria H₂S subsp. thermosyntrophicumAdapted from Henstra et al., Curr. Opin. Biotechnol. 18: 200-206 (2007);Sipma et al., Crit. Rev. Biotechnol. 26: 41-65 (2006)).

One type of organism for consideration of utilizing syngas isthermophilic acetogens due to their ability to tolerate temperatures ashigh as 72° C., which would reduce contamination issues and lower theheating cost associated with separating a product such as butanol viadistillation. However, alcohol production from synthesis gas has yet tobe demonstrated in thermophiles and their primary products are hydrogen,acetate, and/or H₂S. The doubling times of the acetogenic thermophileswere also longer than for mesophilic acetogens. Thus, initial studiesare focused on mesophilic acetogenes for the production of a desiredproduct such as butanol as these organisms have the fastest doublingtimes and have been shown to produce alcohols from syngas. Initialcharacterizations are performed on Clostridium ljungdahlii andClostridium carboxidivorans. Of all syngas-utilizing organisms, C.ljungdahlii has a substantial body of knowledge relating to itsmetabolic capabilities and optimum fermentation conditions. C.carboxidivorans has been shown to naturally produce small quantities ofbutanol during growth on syngas (Henstra et al., Curr. Opin. Biotechnol.18:200-206 (2007)).

The metabolic pathways of some exemplary syngas utilizing organisms areknown. Two exemplary pathways utilizing syngas are shown in FIGS. 1 and2.

Acetogens, such as C. ljungdahlii and C. carboxidivorans, can grow on anumber of carbon sources ranging from hexose sugars to carbon monoxide.Hexoses, such as glucose, are metabolized first viaEmbden-Meyerhof-Parnas (EMP) glycolysis to pyruvate, which is thenconverted to acetyl-CoA via pyruvate:ferredoxin oxidoreductase.Acetyl-CoA can be used to build biomass precursors or can be convertedto acetate, which produces energy via acetate kinase andphosphotransacetylase. The overall conversion of glucose to acetate,energy, and reducing equivalents is:

C₆H₁₂O₆+4ADP+4Pi→2CH₃COOH+2CO₂+4ATP+8[H]

Acetogens extract even more energy out of the glucose to acetateconversion while also maintaining redox balance by further convertingthe CO₂ to acetate via the Wood-Ljungdahl pathway

2CO₂+8[H]+nADP+nPi→CH₃COOH+nATP

The coefficient n in the above equation signify that this conversion isan energy generating endeavor, as many acetogens can grow in thepresence of CO₂ via the Wood-Ljungdahl pathway even in the absence ofglucose as long as hydrogen is present to supply the necessary reducingequivalents.

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

The Wood-Ljungdahl pathway, illustrated in FIG. 3, is coupled to thecreation of Na⁺ or H⁺ ion gradients that can generate ATP via an Na⁺- orH⁺-dependant ATP synthase, respectively (Muller, Appl. Environ.Microbiol. 69:6345-6353 (2003)). Based on these known transformations,acetogens also have the capacity to utilize CO as the sole carbon andenergy source. Specifically, CO can be oxidized to produce reducingequivalents and CO₂, or directly assimilated into acetyl-CoA which issubsequently converted to either biomass or acetate.

4CO+2H₂O→CH₃COOH+2CO₂

Even higher acetate yields, however, can be attained when enoughhydrogen is present to satisfy the requirement for reducing equivalents.

2CO+2H₂→CH₃COOH

Following from FIG. 3, the production of acetate via acetyl-CoAgenerates one ATP molecule, whereas the production of ethanol fromacetyl-CoA does not and requires two reducing equivalents. Thus ethanolproduction from syngas is not expected to generate sufficient energy forcell growth in the absence of acetate production. However, under certainconditions, Clostridium ljungdahlii produces mostly ethanol fromsynthesis gas (Klasson et al., Fuel 72:1673-1678 (1993)), indicatingthat some combination of the following pathways

2CO₂+6H₂→CH₃CH₂OH+3H₂O

6CO+3H₂O→CH₃CH₂OH+4CO₂

2CO+4H₂→CH₃CH₂OH+H₂O

does indeed generate enough energy to support cell growth. Hydrogenicbacteria such as R. rubrum can also generate energy from the conversionof CO and water to hydrogen (see FIG. 3) (Sipma et al., Crit. Rev.Biotechnol. 26:41-65 (2006)). The key mechanism is the coordinatedaction of an energy converting hydrogenase (ECH) and CO dehydrogenase.The CO dehydrogenase supplies electrons from CO which are then used toreduce protons to H₂ by ECH, whose activity is coupled toenergy-generating proton translocation. The net result is the generationof energy via the water-gas shift reaction.

The product profile from syngas fermentations is determined by thechoice of organism and experimental conditions. For example, Clostridiumljungdahlii produces a mixture of ethanol and acetate (Klasson et al.,Fuel 72:1673-1678 (1993); Gaddy and Clausen, U.S. Pat. No. 5,173,429)while Clostridium carboxidivorans produces a mixture of ethanol,acetate, butanol, and butyrate (Liou et al., Int. J. Syst. Evol.Microbiol. 55(Pt 5):2085-2091 (2005)). Acetate and biomassconcentrations as high as 26.8 g/L and 12.4 g/L, respectively, togetherwith ethanol concentrations below 1 g/L have been reported with C.ljungdahlii (Gaddy, U.S. Pat. Nos. 5,807,722, 6,136,577 and 6,340,581).This product profile can be shifted, however, towards increased ethanolformation by traditional means of increasing solvent formation over acidproduction in Clostridia, for example, using nutrient limitation, mediaalteration, lower pH, reducing agent addition, and the like. Productprofile sensitivity to a number of conditions, for example, calciumpantothenate limitation, cobalt limitation, H2 oversupply, COoversupply, acetate conditioning, and the like, has been described(Gaddy et al., U.S. Pat. No. 7,285,402). Ethanol, acetate, and cellconcentrations of 33.0 g/L, 4.0 g/L, and 2.7 g/L, respectively, weredemonstrated with C. ljungdahlii strain C-01 without cell recycle underconditions optimized for ethanol production. Maximum ethanolproductivities ranged from 21 g/L/day without cell recycle to 39 g/L/daywith cell recycle.

Sensitivity of syngas fermentations to inhibitors can also bedetermined. Fewer efforts to optimize the fermentation conditions of C.carboxidivorans (Liou et al., Int. J. Syst. Evol. Microbiol. 55(Pt5):2085-2091 (2005)) for the generation of a particular product havebeen reported. However, a number of recent studies have been aimed atthe inhibition of C. carboxidivorans growth by syngas inhibitors.Specifically, inhibitors present in the biomass-generated producer gasstopped C. carboxidivorans growth and H₂ utilization, although growthcould be recovered when “clean” bottled gasses consisting of only CO,CO₂, N₂, and H₂ were introduced (Datar et al., Biotechnol. Bioeng.86:587-594 (2004)). Passing the gas through a 0.025 μm filter cleaned itwell enough to allow cell growth, although H₂ utilization was stillblocked (Ahmed et al., Biomass Bioenergy 30:665-672 (2006)). A scanningelectron microscope analysis of the filter indicated that tarparticulates, and not ash, was the likely culprit leading to celldormancy. Potential tar species were identified as benzene, toluene,ethylbenzene, p-xylene, o-xylene, and napthalene. Cells were able toadapt to the tars present following the 0.2 μm filter within 10-15 days.The fact that H₂ utilization ceased regardless of filter size indicatedthat a non-filtered component was inhibiting the hydrogenase enzyme.This compound was later identified as nitric oxide. NO inhibitshydrogenase at ≧60 ppm levels (Ahmed and Lewis, Biotechnol. Bioeng.97:1080-1086 (2007)). Similar studies can be performed to determineappropriate conditions for the utilization of syngas in a particularorganism to produce a desired product.

In an exemplary experiment, it is assumed that synthesis gas exiting thegasifier is passed though a cyclone, a condensation tower, a scrubber,and a 0.2 μm filter, similar to the system described previously forswitchgrass gasification (Datar et al., Biotechnol. Bioeng. 86:587-594(2004); Ahmed et al., Biomass Bioenergy 30:665-672 (2006))). Oxygenblown gasification, as opposed to air blown, is used so that NO levelsunder 40 ppm can be achieved, as suggested previously (Ahmed and Lewis,Biotechnol. Bioeng. 97:1080-1086 (2007)). Furthermore, studies with C.ljungdahlii revealed that H₂S levels under 2.7% are not inhibitory(Klasson et al., Fuel 72:1673-1678 (1993)), even when the cells are notacclimated beforehand, and levels are expected to be below that levelwith syngas obtained from biomass or even coal gasification. Inaddition, tolerance to tar particulates can be achieved throughevolution or adaptation (Ahmed et al., Biomass Bioenergy 30:665-67.(2006)).

Example II Design and Modeling of Microbial Strains for Utilization ofSyngas

This example describes the design of exemplary microbial strains for theproduction of a desired product from syngas.

Initial studies utilize genome-scale models of C. ljungdahlii, C.carboxidivorans, and R. rubrum for the design of microbial strainscapable of utilizing syngas as a carbon source. Metabolic models andsimulation algorithms are used to develop strains that utilize syngas.Genomic sequences of desired microorganisms are utilized, along withsequences from closely related species, to construct genome-scalemetabolic models of the target organisms. To facilitate this process,Genomatica has developed a comprehensive methodology to automaticallybuild a first draft of a metabolic network based on an exhaustivesequence comparison with our existing high quality manually builtmetabolic models. Next, the automatically generatedgene-protein-reaction (GPR) assignments, see FIG. 2, are checkedmanually and detailed notes are catalogued within SimPheny™,Genomatica's proprietary model construction and simulation platform, toensure that they are as transparent as possible. For the production ofbutanol as an exemplary product, enzymes in the butanol pathway areexpressed in those organisms that do not produce butanol naturally, forexample, C. ljungdahlii and R. rubrum.

The metabolic models are interrogated using a constraint-based modelingapproach (Schilling et al., Biotechnol. Prog. 15:288-295 (1999); Edwardset al., Environ. Microbiol. 4:133-40 (2002); Varma and Palsson,Biotechnol. 12:994-998 (1994); Patil et al., Curr. Opin. Biotechnol.15:64-69 (2004)). Briefly, rather than attempting to calculate andpredict exactly what an organism does, the constraint-based approachnarrows the range of possible phenotypes that an organism can displaybased on the successive imposition of governing physico-chemicalconstraints, for example, stoichiometric, thermodynamic, capacity, andregulatory (Price et al., Trends Biotechnol. 21:162-169 (2003); Price etal., Nat. Rev. Microbiol. 2:886-897 (2004)). Thus, instead ofcalculating an exact phenotypic “solution,” that is exactly how anorganism will behave under given genetic and environmental conditions,it can determine the feasible set of phenotypic solutions in which theorganism can operate. In general, genome-scale constraint-based modelshave been shown to be useful in predicting several physiologicalproperties such as growth and by-product secretion patterns (Edwards andPalsson, Proc. Natl. Acad. Sci. USA 97:5528-5533 (2000); Varma et al.,Appl. Environ. Microbiol. 59:2465-2473 (1993); Varma and Palsson, ApplEnviron Microbiol, 60:3724-3731 (1994); Edwards et al., Nat. Biotechnol.19:125-130 (2001)), determining the range of substrate utilization(Edwards and Palsson, supra, 2000), determining the minimal media forgrowth (Schilling et al., J Bacteriol. 184:4582-4593 (2002), predictingthe outcome of adaptive evolution Marra et al., Nature 420:186-189(2002)), calculating theoretical product yields (Varma et al.,Biotechnol. Bioengineer. 42:59-73 (1993)), predicting knockoutphenotypes (Edwards and Palsson, BMC Bioinformatics 1:1 (2000); Segre etal., Proc. Natl. Acad. Sci. USA 99:15112-15117 (2002); Shlomi et al.,Proc. Natl. Acad. Sci. USA 102:7695-7700 (2005)) and comparing metaboliccapabilities of different organisms (Forster et al., Genome Res.13:244-253 (2003)). Based on these predictive capabilities, the modelsare used to characterize the metabolic behavior of industrial microbesunder laboratory and production scale fermentation conditions.Constraint-based approaches have matured to the point where they arecommonly applied to pinpoint successful genetic manipulations aimed atimproving strain performance (Bro et al., Metab. Eng. 8:102-111 (2006);Alper et al., Nat. Biotechnol. 23:612-616 (2005); Alper et al., Metab.Eng. 7:155-164 (2005); Fong et al., Biotechnol. Bioeng. 91:643-648(2005); Park et al., Proc. Natl. Acad. Sci. USA 104:7797-7802 (2007)).Characteristics are continued to be monitored in order to implementfurther optimization of conditions.

Additional optimization of organisms can be performed by determininggene knockouts to enhance for production of a desired product, includinggrowth-coupled production of a desired product such as butanol (seeExample V). C. ljundahlii currently can convert mixtures of CO, CO₂, andH₂ to acetate and ethanol, while C. carboxidivorans produces a mixtureof acetate, ethanol, butyrate, and butanol. R. rubrum does not producealcohols naturally, but has been shown to accumulate high levels ofpoly-β-hydroxyalkanoates (PHAs). Modeling analysis allows predictions ofthe effects on cell growth of shifting the metabolism of a biocatalystorganism towards more efficient production of a desired product such asbutanol. The modeling also points at metabolic manipulations aimed atdriving the metabolic flux through a desired production pathway, forexample, the production of butanol. One modeling method is the bileveloptimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in the growth-coupled production of adesired product such as butanol. Strains designed with a gene knockoutstrategy are forced, due to network stoichiometry, to produce highlevels of a desired product such as butanol for efficient growth,because all other growth options have been removed. Such strains areself-optimizing and stable. Accordingly, they typically maintain orimprove upon production levels even in the face of strong growthselective pressures, making them amenable to batch or continuousbioprocessing and also evolutionary engineering.

Several candidate strain are designed and optimization of productionconditions are performed. Fermentation conditions are tested intriplicate, alongside control fermentations using the original processparameters. Using data from test fermentations, simulations can beperformed to assess changes in metabolism that result from processchanges and compared to predictions. If productivity significantly fallsshort of that anticipated, further simulations are performed using thisnew knowledge for a second iteration of the design process in order tooptimize strains.

Example III Development of Genetic Tools for Target Organisms

This example describes the development of tools for genetic manipulationand engineering of target organisms.

Genetic systems are developed in candidate strains for utilization ofsyngas. In particular, genetic systems are developed for C. ljungdahliiand C. carboxidivorans. Genetic transformations are also tested inRhodospirillum rubrum. Antibiotic resistance is tested to determinepotential markers for selection of desired genetic elements. Forexample, many Clostridia are sensitive to erythromycin andchloramphenicol. DNA transfer methods are developed using well knownmethods, including but not limited to electroporation, conjugation orultrasound transformation. Additional testing is performed on severalexpression vectors of gram positive bacteria, particularly the vectorsused in C. acetobutylicum, to determine their effectiveness forexpression of desired genetic elements in C. ljungdahlii and/or C.carboxidivorans. Additional vectors can be developed by replacing thepromoter of the vectors with a native C. ljungdahlii or C.carboxidivorans promoter. In addition, several suicide plasmids,including those of C. acetobutylicum and C. cellulolyticum, are testedfor genetic manipulation. The knockdown technique of antisense RNAinhibition developed for other Clostridia are also tested.

The transformation, expression and antisense RNA inhibition tools areavailable for mesophilic species Clostridium cellulolyticum andClostridium acetobutylicum. C. cellulolyticum is a model system forcellulose degradation (Desvaux, FEMS Microbiol Rev. 741-764 (2005)),whereas C. acetobutylicum has been intensively characterized for itsability to produce solvents such as butanol (Durre, Biotechnol. J.2:1525-1534 (2007)). Notably, both species are capable of producingethanol and hydrogen as an end product. Therefore, knowledge from thesetwo strains is instructive for other ethanol- and/or hydrogen-producingClostridia species. Studies of targeted mutagenesis in C. cellulolyticumhave been initiated and can be similarly used on other candidateorganisms.

The results of these studies allow for phenotypic characterization ofthe mutants generated as well as allow genetic engineering of C.ljungdahlii and/or C. carboxidivorans. Additional optimization isperformed, as needed, to develop genetic systems by varying methods,plasmids and conditions to achieve an optimized result (Lynd et al.,Microbiol. Mol. Biol. Rev. 66:506-577 (2002)).

In more detail, profiling the antibiotic resistance capacities of C.ljungdahlii and C. carboxidivorans is performed. An important step indeveloping genetic systems is to determine the native antibioticresistance characteristics of the target strains. Erythromycin andchloramphenicol are two antibiotics with resistance markers that havebeen shown to be functional on plasmids in C. acetobutylicum and C.cellulolyticum (Kashket and Cao, Appl. Environ. Microbiol. 59:4198-4202(1993); Green and Bennett, Biotechnol. Bioeng. 58:215-221 (1998)).However, they are usually not available for common suicide plasmids,which instead often contain antibiotic markers of ampicillin,gentamycin, rifampicin, kanamycin and tetracycline. In order todetermine antibiotic sensitivity, C. ljungdahlii and C. carboxidivoransare grown in defined medium in an anaerobic chamber (Ahmed and Lewis,Biotechnol. Bioeng. 97:1080-1086 (2007); Younesi et al., Bioresour.Technol. Jun. 18, 2007). Common antibiotics as indicated above are addedat gradient concentrations from 1 μg/ml to 500 μg/ml. An instrument suchas a Type FP-1100-C Bioscreen C machine (Thermo Labsystems; WalthamMass.) is used to control the growth temperature at 37° C. andautomatically measure the optical density of cell growth at differentintervals. All of the physiological studies are performed in replicate,for example, triplicates, so that the average and standard deviation canbe calculated. This growth data indicates the sensitivity of C.ljungdahlii and C. carboxidivorans to the antibiotics being tested. Theantibiotics that inhibit growth of the strain are used in furtherstudies.

In more detail, DNA transfer methods and gene expression systems aredeveloped to provide simple and efficient DNA delivery methods forgenetic engineering. Methods for bacterial DNA transfer includeconjugation, electroporation, chemical transformation, transduction andultrasound transformation. Among them, electroporation and conjugationhave been previously established in several Clostridial species (Jennertet al., Microbiol. 146:3071-3080 (2000); Tardif et al., J. Ind.Microbiol. Biotechnol. 27:271-274 (2001); Tyurin et al., J. Appl.Microbiol. 88:220-227 (2000); Tyurin et al., Appl. Environ. Microbiol.70:883-890 (2004)). Ultrasound transformation is a convenient andefficient method that provides high transformation efficiency (>10⁶CFU/μg DNA) for gram negative bacteria (Song et al., Nucl. Acids Res.35:e129 (2007)) and can be tested in gram positive bacteria.

Electroporation, ultrasound transformation, and conjugation are testedfor C. ljungdahlii and C. carboxidivorans transformation efficiencies. Avariety of plasmids from gram positive bacteria with differentreplicons, for example, pIP404, pAMβ1 and pIM13, are tested. If needed,subcloning is employed to replace the antibiotic resistance cassettes ofexisting plasmids with the suitable ones based on antibiotic resistancetesting. Standard molecular subcloning techniques, including restrictionenzyme digestion, ligation by T4 ligase and E. coli transformation, areused for engineering of the plasmids (Sambrook et al., MolecularCloning: A Laboratory Manual Cold Spring Harbor Laboratory Press(1989)). As required for many other Clostridial species, these plasmidsare methylated prior to DNA delivery to protect them from degradation bythe host bacteria. For electroporation and conjugation, existingprotocols of C. cellulolyticum and C. acetobutylicum are tested first.Parameters such as electroporation setup, recovery time, andconcentration of Ca²⁺ and Mg²⁺ in the electroporation buffer are variedto optimize the transformation efficiency. For ultrasoundtransformation, experiments are conducted under conditions of lowfrequency ultrasound, for example, 40 kHz, and extended recovery time aspreviously described (Song et al., supra, 2007)).

Once an efficient DNA transfer protocol is established for certainplasmids, the plasmids are engineered to incorporate a native C.ljungdahlii or C. carboxidivorans promoter followed by a multiplecloning site to generate expression vectors. It is expected that theexisting expression vectors of C. acetobutylicum, such as pSOS95 andpIMP1, can likely work in C. ljungdahlii or C. carboxidivorans withoutchanges of the promoter, so these plasmids are used for initial testing.

To develop gene disruption methods, several suicide plasmids such aspKNOCK, pDS3.0, pSPUC and pBluescript SKII are screened for suitabilityas suicide plasmids for C. ljungdahlii and/or C. carboxidivorans. Asdiscussed above, if the results either existing antibiotic resistancecassettes are used or are replaced with suitable antibiotic resistancecassettes. A DNA fragment of a selected target gene is subcloned intoappropriate suicide plasmids. The genes selected as the initial targetsare those encoding the alcohol dehydrogenases responsible for ethanolproduction. These genes were selected because they lead to byproductformation, are likely to be identified as targets for disruption forbutanol-producing strains, and provide for an easy screen by analyzingethanol in the fermentation broth. If deletion of an alcoholdehydrogenase in C. carboxidivorans lowers butanol production inaddition to lowering ethanol production due to the broad substratespecificity of these enzymes, an alcohol dehydrogenase which favorsbutanol formation over ethanol formation, such as the adhE2 from C.acetobutylicum (Atsumi et al., Metab. Eng. Sep. 14, 2007), can be clonedalong with the other butanol pathway genes to construct a butanolpathway.

The engineered suicide plasmids are methylated and transferred into C.ljungdahlii and C. carboxidivorans. Colonies are selected on solidmedium containing the appropriate antibiotics. PCR amplification andsubsequent sequencing of the disrupted genomic region, southern blot,and physiological studies are employed to verify the correct disruptionof the targeted gene(s) in the genome. The expression systems can alsobe used as an alternative to gene disruption to express the antisenseRNA of the target gene, which will inhibit but not completely abolishits gene expression. Therefore, the antisense RNA system serves as aconvenient approach of gene knock-down of a desired gene.

Example IV Genetic Assessment of Rhodospirillum rubrum

This example describes development of genetic tools for Rhodospirillumrubrum as an organism for utilization of syngas.

Rhodospirillum rubrum is a Gram negative, purple non-sulfur bacteriumwhich oxidizes CO under anaerobic conditions (Kerby et al., J.Bacteriol. 177:2241-2244 (1995); Kerby et al., J. Bacteriol.174:5284-5294 (1992)). R. rubrum possess a Ni—Fe—S CO dehydrogenase(CODH) that catalyzes the oxidation of CO, which is coupled to theformation of hydrogen (Ensign and Ludden, J. Biol. Chem. 1991.266:18395-18403 (1991)). Given its CO oxidation capacity and ability tofix CO₂ , R. rubrum is capable of efficient growth on syngas in the dark(Do et al., Biotechnol. Bioeng. 97:279-286 (2007)). In addition, it hasbeen shown that during growth on syngas, up to 34% of the total cellularcarbon is stored in the form of poly-β-hydroxyalkanoates (PHA) thatconsist primarily of β-hydroxybutyrate (PHB). The ability of R. rubrumto efficiently direct cellular carbon to form reduced 4-carbon compoundsmake it an attractive platform for engineering production of a desiredproduct such as 1-butanol. In addition, a genetic system has beenestablished for R. rubrum, and a wide range of cloning vectors includingthe broad-host range RK2 derivatives are available (Saegesser et al.,FEMS Microbiol. Lett. 95:7-12 (1992)). Another attractive aspect ofutilizing R. rubrum is that there is considerable overlap in thepathways leading to PHB and 1-butanol synthesis (FIG. 5). Since PHBsynthesis has been studied for its use as a biodegradable plastic,considerable information is available regarding PHB pathway regulationand over expression (Anderson and Dawes, Microbiol. Rev. 54:450-472(1990)). In parallel to establishing the genetic tools necessary formanipulating the Clostridial strains as discussed above, a syntheticoperon consisting of several genes from Clostridium acetobutylicum thatform the 1-butanol synthesis pathway is developed as well.

Since R. rubrum has been sequenced and has a tractable genetic system(Saegesser et al., FEMS Microbiol. Lett. 95:7-12 (1992)), it is expectedthat targeted deletions can be made in selected loci. Broad-host range,site-specific gene excision systems are available which allow markerlessdeletions to be generated (Hoang et al., Gene 212:77-86 (1998)).Therefore, it will be possible to generate multiple knockouts in asingle strain without relying on multiple antibiotic selections. Thismethod can be tested by deleting the PHB synthase gene, which is theterminal step in PHB synthesis (Hustede et al., FEMS Microbiol. Lett.72:285-290 (1992)). This is chosen because PHB synthesis will likelycompete with the proposed butanol pathway for 4-carbon precursors andreducing equivalents (FIG. 5). Successful deletion of the equivalentgene in Methylobacterium extorquens, a gram negative bacterium alsoknown to accumulate over 30% by weight PHB, has been reported with nodeleterious effect on growth (Korotkova and Lidstrom, J. Bacteriol.183:1038-1046 (2001)).

Example V Engineering Microorganisms for Production of Butanol fromSyngas

This example describes engineering microorganisms for production ofbutanol formation from syngas.

In initial studies, Clostridial strains, in particular, C.carboxidivorans, are used to engineer utilization of syngas forproduction of and tolerance to butanol. C. carboxidivorans has beenshown to produce butanol from synthesis gas (Liou et al., Int. J. Syst.Evol. Microbiol. 55(Pt 5):2085-2091 (2005)). C. carboxidivorans isengineered to increase syngas utilization efficiency, increase theefficiency of butanol production from syngas as an exemplary desiredproduct, and to increase product tolerance so that higher yields of adesired product can be obtained.

Preliminary metabolic network analysis has revealed that the theoreticalconversion of lignocellulosic-derived syngas to butanol comparesfavorably to sugar fermentation.

-   -   Syngas to Butanol:

12CO+5H₂O→8ATP+8CO₂+1C₄H₁₀O

4CO+8H₂→4ATP+3H₂O+1C₄H₁₀O

4CO₂+12H₂→2ATP+7H₂O+1C₄H₁₀O

-   -   Sugar to Butanol:

1C₆H₁₂O₆→2ATP+2CO₂+1H₂O+C₄H₁₀O

1.2C₅H₁₀O₅→1.7ATP+2CO₂+1H₂O+C₄H₁₀O

Given that biomass gasification can optimally provide a 1:1 ratio of COto H₂, the production of one mole of butanol will require 12 moles ofCO+H₂. Importantly, the fermentative conversion of syngas to butanol isan energy-generating endeavor, therefore supporting cell growth at highproduct yields. Furthermore, initial calculations reveal the substratecost to be cheaper than the equivalent amount of sugar that would berequired.

As discussed above, models and genetic tools are utilized to designstrains that facilitate the production of butanol or other desiredproducts as an obligatory product of cell growth. In other words, thecell is engineered so that butanol is a necessary electron sink duringgrowth on CO. The strains are constructed, which may have a combinationof gene knockouts and overexpression of appropriate enzymes, and can beevolved for improved production and tolerance of growth conditions.

For construction of Clostridial strains producing butanol, genomeanalysis as discussed above is used to identify biological pathwaysnecessary for establishing and/or improving butanol production in C.ljungdahlii and C. carboxidivorans. Additional improvement in butanolproduction can be achieved by increasing expression of syngasutilization pathway and/or butanol production pathway proteins andenzymes. To express the gene(s) in the targeted biological pathway, geneexpression vectors developed as discussed above are used. If there ismore than one gene, the genes are PCR amplified and cloned into anexpression vector as a synthetic operon. The resulting expressionplasmid is transferred into C. ljungdahlii and C. carboxidivorans.Northern blot and/or real time PCR, or other suitable techniques, areused to examine gene expression at the transcriptional level.

To improve butanol production, it is likely that endogenous gene(s) ofC. ljungdahlii and C. carboxidivorans will be inactivated to reroute theredox potential toward butanol production. To this end, an internal DNAfragment of a targeted gene will be PCR amplified and cloned into asuicide plasmid. Then the plasmid is transferred into C. ljungdahlii andC. carboxidivorans, resulting in disruption of the target gene by singlecrossover recombination. The correct disruption is confirmed bysequencing of the PCR product amplified from the disrupted genomic locusand/or Southern blot, or using other suitable analytical techniques. Ifthere is more than one targeted gene, the suicide plasmids areengineered to change antibiotic marker so that multiple gene knockoutscan be generated in a single strain. It is expected that up to 3 to 6gene deletions may be beneficial in optimizing butanol production.

For genetic engineering of Rhodospirillum rubrum for butanol production,a synthetic operon is developed consisting of several genes that formthe butanol synthesis pathway in Clostridium acetobutylicum. A similarapproach for allowing butanol production in E. coli was recentlyreported, proving that heterologous expression of the pathway in Gramnegative organisms is possible (Atsumi et al., Metab. Eng. Sep. 14,2007). The necessary genes for butanol production in R. rubrum can beexpressed on a broad-host-range expression vector. Expression can becontrolled using an inducible promoter such as the tac promoter. Thesynthetic, 4-gene operon is constructed using a fusion PCR technique andwill include genes for crotonase, butyryl-CoA dehydrogenase, electrontransfer flavoprotein, and aldehyde/alcohol dehydrogenase activities.Fusion/assembly PCR techniques have been used to construct syntheticoperons for expression in heterologous hosts (Craney et al., Nucl. AcidsRes. 35:e46 (2007); Hill et al., Mol. Gen. Genet. 226:41-48 (1991)). Thebutanol operon is transformed into both the wild-type and PHB synthesisdeficient R. rubrum strains, and tested as described below. It is alsopossible that more than one gene is desirable to be targeted for removalbased on modeling studies. These deletions can be implemented using themarkerless method, as discussed above.

As intermediate strains are being constructed, they are testedphysiologically to evaluate progress towards butanol production, as wellas the ability to sustain robust growth and reduced byproduct formation.Initial screening for growth and butanol production is performedinitially in 1 mL microreactors (such as MicroReactor Technologies,Inc.; Mountain View, Calif.). Configurations such as 24-well plates canbe controlled for pH, temperature, and gas composition. As a next step,serum bottles are vigorously shaken in temperature and gas compositioncontrolled incubators. This allows sampling and analysis of the gasheadspace as well as the liquid phase. Products such as butanol,ethanol, and organic acids can be analyzed by gas chromatography (GC/MS)or HPLC using routine procedures. H₂, CO, and CO₂ in the headspace willbe analyzed by GC with Thermal Conductivity Detector (TCD) detectionusing 15% Ar as an internal standard, as described previously (Najafpourand Younesi, Enzyme Microb. Technol. 38:223-228 (2006)). In theseexperiments, synthetic syngas with 1/1 ratio of H₂ in CO is used. Theeffect of gas composition is explored during the fermentationoptimization.

Initially, strains with one or more deletions is analyzed to comparegrowth and fermentation profiles relative to wild-type cells. It ispossible that growth in multiple deletion strains will be poor withoutenhanced expression of the butanol pathway. Strains expressing one ormore butanol pathway genes, or other targets identified by metabolicmodeling, are tested in the wild-type host to assess the ability toenhance flux through the butanol pathway and provide a preliminaryassessment of which steps are likely bottlenecks. Different gene orders,and if possible alternate promoters and ribosome binding sites, aretested to optimize the synthetic operon construct. The construct(s)yielding the most positive results are transformed into the hostcontaining the prescribed gene deletions, and tested as described above.Results are compared to model predictions to assess where unforeseenlimitations and metabolic bottlenecks may exist.

After genetic engineering manipulations are made, adaptive evolution canbe utilized to optimize production in a desired strain. Based on straindesign that couples the production of butanol to growth appliesselection pressure that favors cells with improved growth rate and/oryield and will lead to higher butanol yield. Adaptive evolution istherefore performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling and genetic engineering can be utilized tofurther optimize production. The evolutionary engineering step can becarried out in a device that automatically maintains cells in prolongedexponential growth by the serial passage of batch cultures into freshmedium before the stationary phase is attained. Specifically, when acertain cell density is reached, a fraction of the media withexponentially growing cells is passed from one region to an adjacentregion while fresh media is added for the dilution. By automatingoptical density measurement and liquid handling, serial transfer can beperformed at high rates, thus approaching the efficiency of a chemostatfor evolution of cell fitness (Dykhuizen, Methods Enzymol. 224:613-631(1993)). However, in contrast to a chemostat, which maintains cells in asingle vessel, this procedure eliminates the possibility of detrimentalselection for cells adapted for wall-growth (Chao and Ramsdell, J. Gen.Microbiol. 131:1229-1236 (1985); Lynch et al., Nat. Methods 4:87-93(2007)). In addition, this method allows the cells to be maintained in aclosed system that ensures strict anaerobic conditions, a requirementfor growing the Clostridia.

An additional role that adaptive evolution can play is to developstrains that are more tolerant to butanol and impurities such as NOx andtars. Butanol tolerance levels have not been published for C.ljungdahlii and C. carboxidivorans, and this is measured for wild-typecells to determine tolerated levels. Wild-type C. acetobutylicum hasbeen reported to have a tolerance of approximately 180 mM (1.2% w/v)(Tomas et al., Appl. Environ. Microbiol. 69:4951-4965 (2003)) and havebeen engineered to achieve a tolerance levels as high as 2.1% (Ezeji etal., Chem. Rec. 4:305-314 (2004)). Two approaches are currentlyprevalent to improve the butanol tolerance capacity of Clostridia. Oneinvolves changing the lipid composition and the fluidity of the membranevia rational genetic modification of lipid content, or by evolutionmethods such as serial enrichment (Soucaille et al., Curr. Microbiol.14:295-299 (1987)) or random mutagenesis (Jain et al., U.S. Pat. No.5,192,673 1993). However, tolerance is a complex function of multiplefactors and is difficult to achieve with directed modification alone.Further, the cells are reported to lyse at high concentrations ofbutanol (Van Der Westhuizen et al., Appl. Environ. Microbiol.44:1277-1281 (1982)). Therefore, optimization of strains is based on acombination of genetics, evolution, and metabolic modeling. The wildtype strains can be evolved adaptively in the presence of successivelyincreasing concentrations of butanol to demonstrate that butanoltolerance in Clostridium can be improved through this process. One goalis to optimize cells by evolving the cells to obtain a tolerance ofbutanol, for example, a concentration as high as 25 g/L. A similarprocedure can be performed a similar procedure can be used to evaluatethe tolerance of strains to syngas impurities using, for example, NO andaromatic compounds prevalent in tars. Adaptive evolutions foroptimization of production and/or tolerance of impurities can beperformed sequentially or concurrently. This approach can also beintegrated with directed mutation of genes associated with butanoltolerance and membrane fluidity, to optimize tolerance levels suitablefor commercial scale production.

An exemplary syngas to butanol process is illustrated in FIG. 4. FIG. 4illustrates a block flow diagram for a process of utilizing syngas toproduce butanol.

Example VI Development and Optimization of Syngas Fermentation Processes

This example describes the development and optimization of syngasfermentation processes. A laboratory-scale syngas fermentation usingauthentic syngas is performed to demonstrate and optimize target yieldsfor commercial scale production.

Important process considerations for a syngas fermentation are highbiomass concentration and good gas-liquid mass transfer (Bredwell etal., Biotechnol. Prog. 15:834-844 (1999)). The solubility of CO in wateris somewhat less than that of oxygen. Continuously gas-spargedfermentations can be performed in controlled fermenters with constantoff-gas analysis by mass spectrometry and periodic liquid sampling andanalysis by GC and HPLC. The liquid phase can function in batch mode.Butanol and byproduct formation is measured as a function of time.Although the final industrial process will likely have continuous liquidflow, batch operation can be utilized to study physiology in the earlystages of characterization and optimization. All piping in these systemsare glass or metal to maintain anaerobic conditions. The gas sparging isperformed with glass frits to decrease bubble size and improve masstransfer. Various sparging rates are tested, ranging from about 0.1 to 1vvm (vapor volumes per minute). To obtain accurate measurements of gasuptake rates, periodic challenges are performed in which the gas flow istemporarily stopped, and the gas phase composition is monitored as afunction of time.

Fermentation systems specific for syngas utilization are also developed.Although designs are tested with engineered organisms, testing offermentation systems can be done in parallel to strain development,using wild-type organisms at first. In order to achieve the overalltarget productivity, methods of cell retention or recycle are employed.A usual concern about such systems operated continuously is that cellscould evolve to non-producing phenotypes. Because the organisms aredesigned for growth-coupled production of a desired product, theorganisms are genetically stable. One method to increase the microbialconcentration is to recycle cells via a tangential flow membrane from asidestream. Repeated batch culture can also be used, as previouslydescribed for production of acetate by Moorella (Sakai S., Y.Nakashimada, K. Inokuma, M. Kita, H. Okada, and N. Nishio, Acetate andethanol production from H2 and CO2 by Moorella sp. using a repeatedbatch culture. J. Biosci. Bioeng. 99:252-258 (2005)). Various othermethods can also be used (Bredwell et al., Biotechnol. Prog. 15:834-844(1999); Datar et al., Biotechnol. Bioeng. 86:587-594 (2004)). Additionaloptimization can be tested such as overpressure at 1.5 atm to improvemass transfer (Najafpour and Younesi, Enzyme Microb. Technol. 38:223-228(2006)).

Once satisfactory performance is achieved using pure H₂/CO as the feed,synthetic gas mixtures are generated containing inhibitors likely to bepresent in commercial syngas. For example, a typical impurity profile is4.5% CH₄, 0.1% C₂H₂, 0.35% C₂H₆, 1.4% C₂H₄, and 150 ppm nitric oxide(Datar et al., Biotechnol. Bioeng. 86:587-594 (2004)). Tars, representedby compounds such as benzene, toluene, ethylbenzene, p-xylene, o-xylene,and naphthalene, are added at ppm levels to test for any effect onproduction. For example, it has been shown that 40 ppm NO is inhibitoryto C. carboxidivorans (Ahmed and Lewis, Biotechnol. Bioeng. 97:1080-1086(2007)). Cultures are tested in shake-flask cultures before moving to afermenter. Also, different levels of these potential inhibitorycompounds are tested to quantify the effect they have on cell growth.This knowledge is used to develop specifications for syngas purity,which is utilized for scale up studies and production. If any particularcomponent is found to be difficult to decrease or remove from syngasused for scale up, adaptive evolution procedure can be utilized, asdiscussed above, to adapt cells to tolerate one or more impurities.

Example VII Minimal Gene Sets for Generating Syngas UtilizingMicroorganisms

This example describes determination of a minimal gene/protein sets forgeneration of syngas utilizing microorganisms, particularly in anmicroorganism that does not naturally utilize syngas to produce adesired product.

In general, microorganisms have the ability to generatetetrahydrofolate, and methyl-tetrahydrofolate (Me-THF) is a commonintermediate in biosynthesis, for example, in methionine production.Hence, the Methyl Branch outlined above and shown in FIG. 1 is not aunique feature of organisms that utilize syngas. However, the enzymesrequired for generating Me-THF have been found to be much more active insyngas-utilizing organisms relative to organisms that do not use syngas.In fact, tetrahydrofolate-dependent enzymes from acetogens have 50 to100× higher specific activities than those from other sources such as E.coli and eukaryotes (Morton et al., Genetics and Molecular Biology ofAnaerobic Bacteria, M. Sebald, ed., Chapter 28, pp 389-406,Springer-Verlage, New York, N.Y. (1993)). A more appropriate and uniqueway to define a set of genes/proteins for designing an organism that canutilize syngas is to use the Carbonyl Branch of the pathway (see FIG.2). This branch includes genes for the following six (6) proteins:cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbonmonoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS), acetyl-CoAsynthase disulfide reductase, and a CO-tolerant hydrogenase. Therefore,these six genes/proteins represent a set of one or more proteins forconferring a syngas utilization pathway capable of producing acetyl-CoA.

Example VIII Gene Sets for Generating Syngas Utilizing Microorganisms

This example describes exemplary gene sets for generating syngasutilizing microorganisms.

Formate Dehydrogenase. Formate dehydrogenase is a two subunitselenocysteine-containing protein that catalyzes the incorporation ofCO₂ into formate in Moorella thermoacetica (Andreesen and Ljungdahl, J.Bacteriol. 116:867-873 (1973); Li et al., J. Bacteriol. 92:405-412(1966); Yamamoto et al., J. Biol. Chem. 258:1826-1832 (1983). The loci,Moth_(—)2312 and Moth_(—)2313, are actually one gene that is responsiblefor encoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_(—)2314 (Pierce et al., Environ. Microbiol.(2008)). Another set of genes encoding formate dehydrogenase activitywith a propensity for CO₂ reduction is encoded by Sfum_(—)2703 throughSfum_(—)2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur. J.Biochem. 270:2476-2485 (2003)); Reda et al., Proc. Natl. Acad. Sci. US.A. 105:10654-10658 (2008)). Similar to their M. thermoaceticacounterparts, Sfum_(—)2705 and Sfum_(—)2706 are actually one gene. Asimilar set of genes presumed to carry out the same function are encodedby CHY_(—)0731, CHY_(—)0732, and CHY_(—)0733 in C. hydrogenoformans (Wuet al., PLoS Genet. 1:e65 (2005)).

Protein GenBank ID Organism Moth_2312 YP_431142 Moorella thermoaceticaMoth_2313 YP_431143 Moorella thermoacetica Moth_2314 YP_431144 Moorellathermoacetica Sfum_2703 YP_846816.1 Syntrophobacter fumaroxidansSfum_2704 YP_846817.1 Syntrophobacter fumaroxidans Sfum_2705 YP_846818.1Syntrophobacter fumaroxidans Sfum_2706 YP_846819.1 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 Carboxydothermus hydrogenoformansCHY_0732 YP_359586.1 Carboxydothermus hydrogenoformans CHY_0733YP_359587.1 Carboxydothermus hydrogenoformans

Formyltetrahydrofolate synthetase. Formyltetrahydrofolate synthetaseligates formate to tetrahydrofolate at the expense of one ATP. Thisreaction is catalyzed by the gene product of Moth_(—)0109 in M.thermoacetica (Lovell et al., Arch. Microbiol 149:280-285 (1988); Lovellet al., Biochemistry 29:5687-5694 (1990); O'brien et al., Experientia.Suppl. 26:249-262 (1976), FHS in Clostridium acidurici (Whitehead andRabinowitz, J. Bacteriol. 167:205-209 (1986); Whitehead and Rabinowitz,J. Bacteriol. 170:3255-3261 (1988)), and CHY_(—)2385 in C.hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005)).

Protein GenBank ID Organism Moth_0109 YP_428991.1 Moorella thermoaceticaCHY_2385 YP_361182.1 Carboxydothermus hydrogenoformans FHS P13419.1Clostridium acidurici

Methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase. In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_(—)1516, folD, and CHY_(—)1878, respectively (D'Ari and Rabinowitz,J. Biol. Chem. 266:23953-23958 (1991); Pierce et al., Environ. Microbiol(2008); Wu et al., PLoS Genet. 1:e65 (2005)).

Protein GenBank ID Organism Moth_1516 YP_430368.1 Moorella thermoaceticafolD NP_415062.1 Escherichia coli CHY_1878 YP_360698.1 Carboxydothermushydrogenoformans

Methylenetetrahydrofolate reductase. The final step of the methyl branchof the Wood-Ljungdahl pathway is catalyzed by methylenetetrahydrofolatereductase. In M. thermoacetica, this enzyme is oxygen-sensitive andcontains an iron-sulfur cluster (Clark and Ljungdahl, J Biol. Chem.259:10845-10849 (1984)). This enzyme is encoded by metF in E. coli(Sheppard et al., J. Bacteriol. 181:718-725 (1999)) and CHY_(—)1233 inC. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005)). The M.thermoacetica genes, and its C. hydrogenoformans counterpart, arelocated near the CODH/ACS gene cluster, separated by putativehydrogenase and heterodisulfide reductase genes.

Protein GenBank ID Organism metF NP_418376.1 Escherichia coli CHY_1233YP_360071.1 Carboxydothermus hydrogenoformans

Acetyl-CoA synthase/Carbon monoxide dehydrogenase (ACS/CODH) and relatedproteins. ACS/CODH is the central enzyme of the carbonyl branch of theWood-Ljungdahl pathway. It catalyzes the reversible reduction of carbondioxide to carbon monoxide and also the synthesis of acetyl-CoA fromcarbon monoxide, Coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoide-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionof ACS/CODH in a foreign host involves introducing many, if not all, ofthe following proteins and their corresponding activities.

Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE)

Corrinoid iron-sulfur protein (AcsD)

Nickel-protein assembly protein (AcsF)

Ferredoxin (Orf7)

Acetyl-CoA synthase (AcsB and AcsC)

Carbon monoxide dehydrogenase (AcsA)

Nickel-protein assembly protein (CooC)

The genes required for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Morton et al., J. Biol. Chem. 266:23824-23828(1991); Ragsdale, Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004);Roberts et al., Proc. Natl. Acad. Sci. USA 86:32-36 (1989)). Each of thegenes in this operon from the acetogen, M. thermoacetica, has alreadybeen cloned and expressed actively in E. coli (Lu et al., J. Biol. Chem.268:5605-5614 (1993); Morton et al., supra, 1991; Roberts et al., supra,1989)). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

Protein GenBank ID Organism AcsE YP_430054 Moorella thermoacetica AcsDYP_430055 Moorella thermoacetica AcsF YP_430056 Moorella thermoaceticaOrf7 YP_430057 Moorella thermoacetica AcsC YP_430058 Moorellathermoacetica AcsB YP_430059 Moorella thermoacetica AcsA YP_430060Moorella thermoacetica CooC YP_430061 Moorella thermoacetica

The hydrogenogenic bacterium, Carboxydothermus hydrogenoformans, canutilize carbon monoxide as a growth substrate by means of acetyl-CoAsynthase (Wu et al., PLoS Genet. 1:e65. (2005)). In strain Z-2901, theacetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenasedue to a frameshift mutation (We et al., supra, 2005), whereas in strainDSM 6008, a functional unframeshifted full-length version of thisprotein has been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci.USA 101:446-451 (2004)). The protein sequences of the C.hydrogenoformans genes from strain Z-2901 can be identified by thefollowing GenBank accession numbers. Sequences for Carboxydothermushydrogenoformans DSM 6008 are not currently accessible in publiclyavailable databases but can be readily determined as the sequencesbecome available.

Protein GenBank ID Organism AcsE YP_360065 Carboxydothermushydrogenoformans AcsD YP_360064 Carboxydothermus hydrogenoformans AcsFYP_360063 Carboxydothermus hydrogenoformans Orf7 YP_360062Carboxydothermus hydrogenoformans AcsC YP_360061 Carboxydothermushydrogenoformans AcsB YP_360060 Carboxydothermus hydrogenoformans CooCYP_360059 Carboxydothermus hydrogenoformans

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc. Natl. Acad. Sci. USA 103:17921-17926 (2006)). Thisorganism contains two sets of genes that encode ACS/CODH activity(Rother and Metcalf, Proc. Natl. Acad. Sci. USA 101:16929-16934 (2004)).The protein sequences of both sets of M. acetivorans genes can beidentified by the following GenBank accession numbers.

Protein GenBank ID Organism AcsC NP_618736 Methanosarcina acetivoransAcsD NP_618735 Methanosarcina acetivorans AcsF, CooC NP_618734Methanosarcina acetivorans AcsB NP_618733 Methanosarcina acetivoransAcsEps NP_618732 Methanosarcina acetivorans AcsA NP_618731Methanosarcina acetivorans AcsC NP_615961 Methanosarcina acetivoransAcsD NP_615962 Methanosarcina acetivorans AcsF, CooC NP_615963Methanosarcina acetivorans AcsB NP_615964 Methanosarcina acetivoransAcsEps NP_615965 Methanosarcina acetivorans AcsA NP_615966Methanosarcina acetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (thatis, K_(m), V_(max), k_(cat)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol. 188:463-472(2007)).

In both M. thermoacetica and C. hydrogenoformans, additional CODHencoding genes are located outside of the ACS/CODH operons. Theseenzymes provide the ability to extract electrons, or reducingequivalents, from the conversion of carbon monoxide to carbon dioxide.The reducing equivalents are then passed to acceptors such as oxidizedferredoxin, NADP+, water, or hydrogen peroxide to form reducedferredoxin, NADPH, H₂, or water, respectively. In some cases,hydrogenase encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that is proposed to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO to CO₂ and H₂ (Fox et al., J. Bacteriol. 178:6200-6208(1996)). The CODH-I of C. hydrogenoformans and its adjacent genes havebeen proposed to catalyze a similar functional role based on theirsimilarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al.,PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shownto exhibit intense CO oxidation and CO₂ reduction activities when linkedto an electrode (Parkin et al., J. Am. Chem. Soc. 129:10328-10329(2007)). The genes encoding the C. hydrogenoformans CODH-II and CooF, aneighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMSMicrobiol. Lett. 191:243-247 (2000)). The resulting complex wasmembrane-bound, although cytoplasmic fractions of CODH-II were shown tocatalyze the formation of NADPH suggesting an anabolic role(Svetlitchnyi et al., J. Bacteriol. 183:5134-5144 (2001)). The crystalstructure of the CODH-II is also available (Dobbek et al., Science293:1281-1285 (2001)). The protein sequences of exemplary CODH andhydrogenase genes can be identified by the following GenBank accessionnumbers.

Protein GenBank ID Organism CODH (putative) YP_430813 Moorellathermoacetica CODH-I (CooS-I) YP_360644 Carboxydothermushydrogenoformans CooF YP_360645 Carboxydothermus hydrogenoformans HypAYP_360646 Carboxydothermus hydrogenoformans CooH YP_360647Carboxydothermus hydrogenoformans CooU YP_360648 Carboxydothermushydrogenoformans CooX YP_360649 Carboxydothermus hydrogenoformans CooLYP_360650 Carboxydothermus hydrogenoformans CooK YP_360651Carboxydothermus hydrogenoformans CooM YP_360652 Carboxydothermushydrogenoformans CooM AAC45116 Rhodospirillum rubrum CooK AAC45117Rhodospirillum rubrum CooL AAC45118 Rhodospirillum rubrum CooX AAC45119Rhodospirillum rubrum CooU AAC45120 Rhodospirillum rubrum CooH AAC45121Rhodospirillum rubrum CooF AAC45122 Rhodospirillum rubrum CODH (CooS)AAC45123 Rhodospirillum rubrum CooC AAC45124 Rhodospirillum rubrum CooTAAC45125 Rhodospirillum rubrum CooJ AAC45126 Rhodospirillum rubrumCODH-II YP_358957 Carboxydothermus hydrogenoformans (CooS-II) CooFYP_358958 Carboxydothermus hydrogenoformans

Acetyl-CoA synthase disulfide reductase. In Moorella thermoacetica, aset of genes encoding a heterodisulfide reductase (Moth_(—)1194 toMoth_(—)1196) is located directly downstream of the acs gene clusterdiscussed above. In addition, like M. thermoacetica, C. hydrogenoformanscontains a set of genes encoding heterodisulfide reductase directlyfollowing acsE.

Protein GenBank ID Organism HdrC YP_430053 Moorella thermoacetica HdrBYP_430052 Moorella thermoacetica HdrA YP_430052 Moorella thermoaceticaHdrC YP_360066 Carboxydothermus hydrogenoformans HdrB YP_360067Carboxydothermus hydrogenoformans HdrA YP_360068 Carboxydothermushydrogenoformans

Hydrogenase (Hyd). Unlike the redox neutral conversion of CO andmethanol to acetyl-CoA or acetate, the production of more highly reducedproducts such as ethanol, butanol, isobutanol, isopropanol,1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, methacrylicacid, adipic acid, and acrylic acid at the highest possible yieldrequires the extraction of additional reducing equivalents from both COand H₂ (for example, see ethanol formation in FIG. 7). Specifically,reducing equivalents (for example, 2[H] in FIG. 6) are obtained by theconversion of CO and water to CO₂ via carbon monoxide dehydrogenase asdescribed in Example II or directly from the activity of ahydrogen-utilizing hydrogenase which transfers electrons from H₂ to anacceptor such as ferredoxin, flavodoxin, FAD+, NAD+, or NADP+.

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J. Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J. Biochem. 156:265-275 (1986); Sawers et al., J. Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities, it ispossible that E. coli or another host organism can provide sufficienthydrogenase activity to split incoming molecular hydrogen and reduce thecorresponding acceptor. Among the endogenous hydrogen-lyase enzymes ofE. coli are hydrogenase 3, a membrane-bound enzyme complex usingferredoxin as an acceptor, and hydrogenase 4, which also uses aferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyfgene clusters, respectively. Hydrogenase activity in E. coli is alsodependent upon the expression of the hyp genes whose correspondingproteins are involved in the assembly of the hydrogenase complexes(Jacobi et al., Arch. Microbiol. 158:444-451 (1992); Rangarajan et al.,J. Bacteriol. 190:1447-1458 (2008)). The M. thermoacetica hydrogenasesare suitable candidates should the production host lack sufficientendogenous hydrogenase activity. M. thermoacetica can grow with CO₂ asthe exclusive carbon source, indicating that reducing equivalents areextracted from H₂ to allow acetyl-CoA synthesis via the Wood-Ljungdahlpathway (Drake, J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res.Microbiol. 155:869-883 (2004); Kellum and Drake, J. Bacteriol.160:466-469 (1984)) (see FIG. 6). M. thermoacetica has homologs toseveral hyp, hyc, and hyf genes from E. coli. These protein sequencesencoded for by these genes can be identified by the following GenBankaccession numbers. In addition, several gene clusters encodinghydrogenase and/or heterodisulfide reductase functionality are presentin M. thermoacetica and their corresponding protein sequences are alsoprovided below.

Hyp assembly proteins.

Protein GenBank ID Organism HypA NP_417206 Escherichia coli HypBNP_417207 Escherichia coli HypC NP_417208 Escherichia coli HypDNP_417209 Escherichia coli HypE NP_417210 Escherichia coli HypFNP_417192 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes.

Protein GenBank ID Organism Moth_2175 YP_431007 Moorella thermoaceticaMoth_2176 YP_431008 Moorella thermoacetica Moth_2177 YP_431009 Moorellathermoacetica Moth_2178 YP_431010 Moorella thermoacetica Moth_2179YP_431011 Moorella thermoacetica Moth_2180 YP_431012 Moorellathermoacetica Moth_2181 YP_431013 Moorella thermoacetica

Hydrogenase 3.

Protein GenBank ID Organism HycA NP_417205 Escherichia coli HycBNP_417204 Escherichia coli HycC NP_417203 Escherichia coli HycDNP_417202 Escherichia coli HycE NP_417201 Escherichia coli HycFNP_417200 Escherichia coli HycG NP_417199 Escherichia coli HycHNP_417198 Escherichia coli HycI NP_417197 Escherichia coli

Hydrogenase 4.

Protein GenBank ID Organism HyfA NP_416976 Escherichia coli HyfBNP_416977 Escherichia coli HyfC NP_416978 Escherichia coli HyfDNP_416979 Escherichia coli HyfE NP_416980 Escherichia coli HyfFNP_416981 Escherichia coli HyfG NP_416982 Escherichia coli HyfHNP_416983 Escherichia coli HyfI NP_416984 Escherichia coli HyfJNP_416985 Escherichia coli HyfR NP_416986 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyc and/or hyf genes.

Protein GenBank ID Organism Moth_2182 YP_431014 Moorella thermoaceticaMoth_2183 YP_431015 Moorella thermoacetica Moth_2184 YP_431016 Moorellathermoacetica Moth_2185 YP_431017 Moorella thermoacetica Moth_2186YP_431018 Moorella thermoacetica Moth_2187 YP_431019 Moorellathermoacetica Moth_2188 YP_431020 Moorella thermoacetica Moth_2189YP_431021 Moorella thermoacetica Moth_2190 YP_431022 Moorellathermoacetica Moth_2191 YP_431023 Moorella thermoacetica Moth_2192YP_431024 Moorella thermoacetica

Additional hydrogenase-encoding gene clusters in M. thermoacetica.

Protein GenBank ID Organism Moth_0439 YP_429313 Moorella thermoaceticaMoth_0440 YP_429314 Moorella thermoacetica Moth_0441 YP_429315 Moorellathermoacetica Moth_0442 YP_429316 Moorella thermoacetica Moth_0809YP_429670 Moorella thermoacetica Moth_0810 YP_429671 Moorellathermoacetica Moth_0811 YP_429672 Moorella thermoacetica Moth_0812YP_429673 Moorella thermoacetica Moth_0813 (possible psuedogene,Moorella thermoacetica GenBank ID unavailable) Moth_0814 YP_429674Moorella thermoacetica Moth_0815 YP_429675 Moorella thermoaceticaMoth_0816 YP_429676 Moorella thermoacetica Moth_1193 YP_430050 Moorellathermoacetica Moth_1194 YP_430051 Moorella thermoacetica Moth_1195YP_430052 Moorella thermoacetica Moth_1196 YP_430053 Moorellathermoacetica Moth_1717 YP_430562 Moorella thermoacetica Moth_1718YP_430563 Moorella thermoacetica Moth_1719 YP_430564 Moorellathermoacetica Moth_1883 YP_430726 Moorella thermoacetica Moth_1884YP_430727 Moorella thermoacetica Moth_1885 YP_430728 Moorellathermoacetica Moth_1886 YP_430729 Moorella thermoacetica Moth_1887YP_430730 Moorella thermoacetica Moth_1888 YP_430731 Moorellathermoacetica Moth_1452 YP_430305 Moorella thermoacetica Moth_1453YP_430306 Moorella thermoacetica Moth_1454 YP_430307 Moorellathermoacetica

A host organism engineered with these capabilities that also naturallypossesses the capability for anapleurosis (for example, E. coli) canpotentially grow more efficiently on the syngas-generated acetyl-CoA inthe presence of a suitable external electron acceptor such as nitrate.This electron acceptor is required to accept electrons from the reducedquinone formed via succinate dehydrogenase. A further advantage ofadding an external electron acceptor is that additional energy for cellgrowth, maintenance, and product formation can be generated fromrespiration of acetyl-CoA. An alternative strategy involves engineeringa pyruvate ferredoxin oxidoreductase (PFOR) enzyme into the strain toallow synthesis of biomass precursors in the absence of an externalelectron acceptor.

Pyruvate ferredoxin oxidoreductase (PFOR). Anaerobic growth on synthesisgas and methanol in the absence of an external electron acceptor isconferred upon the host organism with ACS/CODH activity by allowingpyruvate synthesis via pyruvate ferredoxin oxidoreductase (PFOR). ThePFOR from Desulfovibrio africanus has been cloned and expressed in E.coli, resulting in an active recombinant enzyme that was stable forseveral days in the presence of oxygen (Pieulle et al., J. Bacteriol.179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORsand is believed to be conferred by a 60 residue extension in thepolypeptide chain of the D. africanus enzyme. The M. thermoacetica PFORis also well characterized (Menon and Ragsdale, Biochemistry36:8484-8494 (1997)) and was shown to have high activity in thedirection of pyruvate synthesis during autotrophic growth (Furdui andRagsdale, J. Biol. Chem. 275:28494-28499 (2000)). Further, E. colipossesses an uncharacterized open reading frame, ydbK, that encodes aprotein that is 51% identical to the M. thermoacetica PFOR. Evidence forpyruvate oxidoreductase activity in E. coli has been described(Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)). The proteinsequences of these exemplary PFOR enzymes can be identified by thefollowing GenBank accession numbers. Several additional PFOR enzymeshave been described (Ragsdale, Chem. Rev. 103:2333-2346 (2003)).

Protein GenBank ID Organism Por CAA70873.1 Desulfovibrio africanus PorYP_428946.1 Moorella thermoacetica YdbK NP_415896.1 Escherichia coli

This example describes exemplary gene sets for engineering an organismto produce acetyl-CoA from gasses comprising at least one of CO, CO₂,and H₂.

Example IX Engineering a Syngas Utilization Pathway into a Microorganism

This example describes engineering a microorganism to contain a syngasutilization pathway.

In addition to improving the efficiency of microorganisms such asClostridial species that have the natural ability to utilize CO and/orCO₂ as a carbon source (Examples II, III and V), microorganisms that donot have the natural ability to utilize CO and/or CO₂ are engineered toexpress one or more proteins or enzymes that confer a CO and/or CO₂utilization pathway. One exemplary pathway is the Wood-Ljungdahlpathway, which allows the utilization of CO and/or CO₂ as a carbonsource, thereby allowing the microorganism to utilize syngas or othergaseous carbon source (see Examples I and VII).

In initial studies, Escherichia coli, which does not utilize syngasnaturally, is used as a target organism to introduce a CO and/or CO₂utilization pathway such as the Wood-Ljungdahl pathway. TheWood-Ljungdahl pathway involves oxygen sensitive and membrane boundproteins as well as specific co-factors that are not native in E. coli.While several Wood-Ljungdahl pathway genes have been cloned into E.coli, only one enzyme, methyltransferase, was found be expressed inactive form (Roberts et al., Proc. Natl. Acad. Sci. USA 86:32-36(1989)). Purification of the carbonyl branch (see FIG. 2) pathway genesfrom Clostridium thermoaceticum revealed the minimum set of enzymesrequired for in vitro conversion of methyl-THF to acetyl-CoA studies(Roberts et al., J. Bacteriol. 174:4667-4676 (1992)).

Initial studies are directed to engineering a Wood-Ljungdahl pathway, inparticular the carbonyl branch (FIG. 2), into E. coli and testing growthand acetate production from both methyl-THF and syngas. E. coli providesa good model for developing a non-naturally occurring microorganismcapable of utilizing syngas or other gaseous carbon sources since it isamenable to genetic manipulation and is known to be capable of producingvarious products like ethanol, acetate, and succinate effectively underanaerobic conditions from glucose.

To generate an E. coli strain engineered to contain a Wood-Ljungdahlpathway, nucleic acids encoding proteins and enzymes required for thecarbonyl branch of the pathway (see FIG. 2 and Example VII) areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al.,supra, 1989). As described previously, the gene cluster encoding keyproteins in acetyl-CoA synthesis in Clostridium thermoaceticum has beencloned and expressed in E. coli (Roberts et al., supra, 1989). Specificvariation of conditions, such as metal composition of the medium, isrequired to ensure production of active proteins. Genes encodingcobalamide corrinoid/iron-sulfur protein, methyltransferase, carbonmonoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS), acetyl-CoAsynthase disulfide reductase, and a CO-tolerant hydrogenase are clonedand expressed in E. coli to introduce carbonyl branch enzymes of theWood-Ljungdahl pathway (for Wood-Ljungdahl pathway genes, see alsoRagsdale, Critical Rev. Biochem. Mol. Biol. 39:165-195 (2004)). Since E.coli does not normally synthesize cobalamin or cobalamin-like cofactors,which is required for the cobalamide-corrinoid/iron sulfur proteinactivity, the cofactors or genes encoding proteins and enzymes forsynthesis of the required cofactors can also be introduced. Thecobalamin or cobalamin-like cofactors can be provided to the medium,although cost would possibly prohibit this approach for scale up andcommercial manufacture. A better alternative is to clone and express therequisite genes in the E. coli strain expressing the cobalamin-requiringproteins. This has been demonstrated by transfer and functionalexpression of a cobalamin operon containing 20 genes from Salmonellatyphimurium into E. coli (Raux et al., J. Bacteriol. 178:753-767(1996)).

The expression of Wood-Ljungdahl pathway genes is tested using routineassays for determining the expression of introduced genes, for example,Northern blots, PCR amplification of mRNA, immunoblotting, or other wellknown assays to confirm nucleic acid and protein expression ofintroduced genes. Enzymatic activity of the expressed enzymes can betested individually or for production of a product such as acetyl-CoA(see, for example, Roberts et al., supra, 1989). The ability of theengineered E. coli strain to utilize CO and/or CO₂ as a carbon source toproduce acetyl-CoA can be analyzed directly using gaschromatography-mass spectrometry (GCMS) or liquid chromatography-massspectrometry (LCMS), or through the use of metabolic radioactive orisotopic labeling, for example, with radioactive CO or CO₂ and analysisof incorporation of radioactive label into the acetyl-CoA product orincorporation of an isotopically labeled CO or CO₂ precursor andanalysis by techniques such as mass spectrometry (GCMS or LCMS) ornuclear magnetic resonance spectroscopy (NMR). Growth of E. coli usingonly CO and/or CO₂ as a sole carbon source, with or without the presenceof H₂, is another useful test for a fully functional pathway.

Once a functional Wood-Ljungdahl pathway has been engineered into an E.coli strain, the strain is optimized for efficient utilization of thepathway. The engineered strain can be tested to determine if any of theintroduced genes are expressed at a level that is rate limiting. Asneeded, increased expression of one or more proteins or enzymes that maylimit the flux through the pathway can be used to optimize utilizationof the pathway and production of acetyl-CoA.

Metabolic modeling can be utilized to optimize growth conditions (seeExample II). Modeling can also be used to design gene knockouts thatadditionally optimize utilization of the pathway (see Examples II, IVand V and, for example, U.S. patent publications US 2002/0012939, US2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379).Modeling analysis allows predictions of the effects on cell growth ofshifting the metabolism towards more efficient production of acetyl-CoAor other desired product. One modeling method is the bileveloptimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in the growth-coupled production ofacetyl-CoA or other desired products, as discussed below. Strainsdesigned with a gene knockout strategy are forced, due to networkstoichiometry, to produce high levels of a desired product for efficientgrowth, because all other growth options have been removed. Such strainsare self-optimizing and stable. Accordingly, they typically maintain orimprove upon production levels even in the face of strong growthselective pressures, making them amenable to batch or continuousbioprocessing and also evolutionary engineering. Adaptive evolution canbe used to further optimize the production of acetyl-CoA (see ExampleV). Adaptive evolution is therefore performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be utilized to further optimize production and toleranceof enzymes to syngas or impurities in syngas.

Once an engineered microbial strain has been optimized for utilizationof the Wood-Ljungdahl pathway, optimization of the fermentation processcan be performed to increase yields using well known methods and asdescribed, for example, in Example VI). For example, a productivitylevel of 20 g/L acetate at 0.5 g/L/h from syngas would represent adesirable production range towards which further optimization of thestrain for efficient utilization of the pathway as well as optimizationof fermentation conditions can be employed to achieve a desiredproduction level.

Although exemplified with introduction of the carbonyl branch to conferthe ability to utilize CO and/or CO₂ to an engineered microbial strain,a similar approach is applied to introduce enzymes for production ofmethyl-THF to E. coli. As discussed above in Example VII, E. coli hasthe ability to produce methyl-THF, but THF-dependent enzymes fromacetogens have higher specific activities (Morton et al., supra, 1993).Using methods as described above to introduce the carbonyl branch of theWood-Ljungdahl pathway, methyl branch enzymes are introduced into E.coli using similar techniques. Genes encoding one or more of the enzymesferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase are introduced (see FIG. 1). In this case, the genes areintroduced to increase an endogenous enzyme activity and/or to increasethe efficiency of utilization of CO and/or CO₂ to produce methyl-THF.Optimization of the pathway and fermentation conditions is carried outas described above. In addition, both the carbonyl and methyl branchesof the Wood-Ljungdahl pathway can be introduced into the samemicroorganism. In such an engineered organism, the increased productionof methyl-THF from CO and/or CO₂ can be utilized to further increase theproduction of acetyl-CoA in an organism engineered to utilize CO and/orCO₂ using the carbonyl branch of the Wood-Ljungdahl pathway (see FIGS. 3and 6).

Acetyl-CoA can function as a precursor for other desired products. Oncethe acetyl-CoA-producing microorganism has been generated, additionalgenes can be introduced into the microorganism to utilize acetyl-CoA asa precursor to produce other desired products from CO and/or CO₂ ascarbon source. For example, enzymes for butanol production can beintroduced (see FIG. 3 and Example V). Representative genes for butanolpathway from acetyl-CoA are: AtoB, acetyl-CoA acetyltransferase; Th1,acetyl-CoA thiolase; Hbd, 3-hydroxbutyrl-CoA dehydrogenase; Crt,crotonase; Bcd, butyryl-CoA dehydrogenase; Etf, electron transferflavoprotein; AdhE2, aldehyde/alcohol dehydrogenase (see Atsumi et al.,Metabolic Engineering Sep. 14, 2007).

Metabolic pathways for production of additional desired products,including succinate, 4-hydroxybutyrate and 1,4-butanediol are described,for example, in U.S. application Ser. No. 11/891,602, filed Aug. 10,2007, and WO/2008/115840 and enzymes for such pathways can similarly beintroduced, for example, succinyl-CoA ligase, succinyl-CoA:CoAtransferase, succinate semialdehyde dehydrogenase, 4-hydroxybutyric aciddehydrogenase, glutamate:succinic semialdehyde transaminase,4-hydroxybutyryl-CoA transferase, a CoA-dependent aldehydedehydrogenase, alcohol dehydrogenase, and the like. Acetyl-CoA feedsdirectly into the TCA cycle of all cells and succinate is a TCA cycleintermediate. Thus, additional enzymes conferring pathways capable ofutilizing acetyl-CoA produced from CO and/or CO₂ can be engineered andoptimized, as described above, to produce a desired product from theengineered microorganism.

Example X Pathways for the Production of Acetyl-CoA from Synthesis Gasand Methanol

This example describes exemplary pathways for utilization of synthesisgas (syngas) and methanol to produce acetyl-CoA.

An organism capable of producing acetyl-CoA from syngas and methanolcontains two key capabilities, which are depicted in FIG. 7. Onecapability is a functional methyltransferase system that allows theproduction of 5-methyl-tetrahydrofolate (Me-THF) from methanol and THF.A second capability is the ability to combine CO, Coenzyme A, and themethyl group of Me-THF to form acetyl-CoA. The organism is able to ‘fix’carbon from exogenous CO and/or CO₂ and methanol to synthesizeacetyl-CoA, cell mass, and products. This pathway to form acetyl-CoAfrom methanol and syngas is energetically advantageous compared toutilizing the full Wood-Ljungdahl pathway. For example, the directconversion of synthesis gas to acetate is an energetically neutralprocess (see FIG. 6). Specifically, one ATP molecule is consumed duringthe formation of formyl-THF by formyl-THF synthase, and one ATP moleculeis produced during the production of acetate via acetate kinase. Thisnew strategy involving methanol circumvents the ATP consumptionrequirement by ensuring that the methyl group on the methyl branchproduct, methyl-THF, is obtained from methanol rather than CO₂. Thisthereby ensures that acetate formation has a positive ATP yield that canhelp support cell growth and maintenance. A host organism engineeredwith these capabilities that also naturally possesses the capability foranapleurosis (for example, E. coli) can grow on the methanol andsyngas-generated acetyl-CoA in the presence of a suitable externalelectron acceptor such as nitrate. This electron acceptor is required toaccept electrons from the reduced quinone formed via succinatedehydrogenase. A further advantage of adding an external electronacceptor is that additional energy for cell growth, maintenance, andproduct formation can be generated from respiration of acetyl-CoA.

An alternative strategy involves engineering a pyruvate ferredoxinoxidoreductase (PFOR) enzyme into the strain to allow synthesis ofbiomass precursors in the absence of an external electron acceptor. Afurther characteristic of the engineered organism is the capability forextracting reducing equivalents from molecular hydrogen. This allows ahigh yield of reduced products such as ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, methacrylicacid, adipic acid, and acrylic acid.

The organisms can produce acetyl-CoA, cell mass, and targeted chemicalsfrom the following sources: 1) methanol and CO, 2) methanol, CO₂, andH₂, 3) methanol, CO, CO₂, and H₂, 4) methanol and synthesis gascomprising CO and H₂, and 5) methanol and synthesis gas comprising CO,CO₂, and H₂.

Successfully engineering this pathway into an organism involvesidentifying an appropriate set of enzymes, cloning their correspondinggenes into a production host, optimizing the stability and expression ofthese genes, optimizing fermentation conditions, and assaying forproduct formation following fermentation (see Examples II-IV). Describedbelow are a number of enzymes that catalyze each step of the pathwayrequired for the conversion of synthesis gas and methanol to acetyl-CoA.To engineer a production host for the utilization of syngas andmethanol, one or more exogenous DNA sequence(s) encoding the requisiteenzymes are expressed in the microorganism.

This example describes exemplary pathways for acetyl-CoA production fromsyngas and methanol.

Example XI Gene Sets for Generating Methanol and Syngas UtilizingMicroorganisms

This example describes exemplary gene sets for generating methanol andsyngas utilizing microorganisms.

Methanol-methyltransferase (MTR). Expression of the modifiedWood-Ljungdahl pathway in a foreign host (see FIG. 7) requiresintroducing a set of methyltransferases to utilize the carbon andhydrogen provided by methanol and the carbon provided by CO and/or CO₂.A complex of 3 methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired methanol methyltransferase activity (Naidu andRagsdale, J. Bacteriol. 183:3276-3281 (2001); Ragsdale, Crit. Rev.Biochem. Mol. Biol. 39:165-195 (2004); Sauer et al., Eur. J. Biochem.243:670-677 (1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301(1996); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallantet al., J. Biol. Chem. 276:4485-4493 (2001)).

Methanol methyltransferase (MtaB) and Corrinoid protein (MtaC). MtaB isa zinc protein that catalyzes the transfer of a methyl group frommethanol to MtaC, a corrinoid protein. Exemplary genes encoding MtaB andMtaC can be found in methanogenic archaea such as Methanosarcina barkeri(Maeder et al., J. Bacteriol. 188:7922-7931 (2006)) and Methanosarcinaacetivorans (Galagan et al., Genome Res. 12:532-542 (2002)), as well asthe acetogen, Moorella thermoacetica (Das et al., Proteins 67:167-176(2007)). In general, the MtaB and MtaC genes are adjacent to one anotheron the chromosome as their activities are tightly interdependent. Theprotein sequences of various MtaB and MtaC encoding genes in M. barkeri,M. acetivorans, and M. thermoaceticum can be identified by theirfollowing GenBank accession numbers.

Protein GenBank ID Organism MtaB1 YP_304299 Methanosarcina barkeri MtaC1YP_304298 Methanosarcina barkeri MtaB2 YP_307082 Methanosarcina barkeriMtaC2 YP_307081 Methanosarcina barkeri MtaB3 YP_304612 Methanosarcinabarkeri MtaC3 YP_304611 Methanosarcina barkeri MtaB1 NP_615421Methanosarcina acetivorans MtaB1 NP_615422 Methanosarcina acetivoransMtaB2 NP_619254 Methanosarcina acetivorans MtaC2 NP_619253Methanosarcina acetivorans MtaB3 NP_616549 Methanosarcina acetivoransMtaC3 NP_616550 Methanosarcina acetivorans MtaB YP_430066 Moorellathermoacetica MtaC YP_430065 Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_(—)304299 and YP_(—)304298, from M.barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. J.Biochem. 243:670-677 (1997)). The crystal structure of thismethanol-cobalamin methyltransferase complex is also available(Hagemeier et al., Proc. Natl. Acad. Sci. USA 103:18917-18922 (2006)).The MtaB genes, YP_(—)307082 and YP_(—)304612, in M. barkeri wereidentified by sequence homology to YP_(—)304299. In general, homologysearches are an effective means of identifying methanolmethyltransferases because MtaB encoding genes show little or nosimilarity to methyltransferases that act on alternative substrates suchas trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide.The MtaC genes, YP_(—)307081 and YP_(—)304611, were identified based ontheir proximity to the MtaB genes and also their homology toYP_(—)304298. The three sets of MtaB and MtaC genes from M. acetivoranshave been genetically, physiologically, and biochemically characterized(Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194 (2005)). Mutantstrains lacking two of the sets were able to grow on methanol, whereas astrain lacking all three sets of MtaB and MtaC genes sets could not growon methanol. This suggests that each set of genes plays a role inmethanol utilization. The M. thermoacetica MtaB gene was identifiedbased on homology to the methanogenic MtaB genes and also by itsadjacent chromosomal proximity to the methanol-induced corrinoidprotein, MtaC, which has been crystallized (Zhou et al., ActaCrystallogr. Sect. F Struct. Biol. Cryst. Commun. 61:537-540 (2005)) andfurther characterized by Northern hybridization and Western blotting(Das et al., Proteins 67:167-176 (2007)).

Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA). MtaAis zinc protein that catalyzes the transfer of the methyl group fromMtaC either to Coenzyme M in methanogens or to tetrahydrofolate inacetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

Protein GenBank ID Organism MtaA YP_304602 Methanosarcina barkeri MtaA1NP_619241 Methanosarcina acetivorans MtaA2 NP_616548 Methanosarcinaacetivorans

The MtaA gene, YP_(—)304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al., J.Bacteriol. 190:4017-4026 (2008)). It is also important to note thatthere are multiple additional MtaA homologs in M. barkeri and M.acetivorans that are as yet uncharacterized, but may also catalyzecorrinoid protein methyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_(—)304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to tetrahydrofolate given the similar roles oftetrahydrofolate and Coenzyme M in methanogens and acetogens,respectively. The protein sequences of putative MtaA encoding genes fromM. thermoacetica can be identified by the following GenBank accessionnumbers.

Protein GenBank ID Organism MtaA YP_430937 Moorella thermoacetica MtaAYP_431175 Moorella thermoacetica MtaA YP_430935 Moorella thermoacetica

Acetyl-CoA synthase/Carbon monoxide dehydrogenase (ACS/CODH). ACS/CODHis the central enzyme of the carbonyl branch of the Wood-Ljungdahlpathway. It catalyzes the reversible reduction of carbon dioxide tocarbon monoxide and also the synthesis of acetyl-CoA from carbonmonoxide, Coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoide-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionof ACS/CODH in a foreign host involves introducing many, if not all, ofthe following proteins and their corresponding activities.

Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE)

Corrinoid iron-sulfur protein (AcsD)

Nickel-protein assembly protein (AcsF)

Ferredoxin (Orf7)

Acetyl-CoA synthase (AcsB and AcsC)

Carbon monoxide dehydrogenase (AcsA)

Nickel-protein assembly protein (CooC)

The genes required for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Morton et al., J. Biol. Chem. 266:23824-23828(1991); Ragsdale, Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004);Roberts et al., Proc. Natl. Acad. Sci. USA 86:32-36 (1989)). Each of thegenes in this operon from the acetogen, M. thermoacetica, has alreadybeen cloned and expressed actively in E. coli (Lu et al., J. Biol. Chem.268:5605-5614 (1993); Morton et al., supra, 1991; Roberts et al., supra,1989)). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

Protein GenBank ID Organism AcsE YP_430054 Moorella thermoacetica AcsDYP_430055 Moorella thermoacetica AcsF YP_430056 Moorella thermoaceticaOrf7 YP_430057 Moorella thermoacetica AcsC YP_430058 Moorellathermoacetica AcsB YP_430059 Moorella thermoacetica AcsA YP_430060Moorella thermoacetica CooC YP_430061 Moorella thermoacetica

The hydrogenogenic bacterium, Carboxydothermus hydrogenoformans, canutilize carbon monoxide as a growth substrate by means of acetyl-CoAsynthase (Wu et al., PLoS Genet. 1:e65. (2005)). In strain Z-2901, theacetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenasedue to a frameshift mutation (We et al., supra, 2005), whereas in strainDSM 6008, a functional unframeshifted full-length version of thisprotein has been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci.USA 101:446-451 (2004)). The protein sequences of the C.hydrogenoformans genes from strain Z-2901 can be identified by thefollowing GenBank accession numbers. Sequences for Carboxydothermushydrogenoformans DSM 6008 are not currently accessible in publiclyavailable databases but can be readily determined as the sequencesbecome available.

Protein GenBank ID Organism AcsE YP_360065 Carboxydothermushydrogenoformans AcsD YP_360064 Carboxydothermus hydrogenoformans AcsFYP_360063 Carboxydothermus hydrogenoformans Orf7 YP_360062Carboxydothermus hydrogenoformans AcsC YP_360061 Carboxydothermushydrogenoformans AcsB YP_360060 Carboxydothermus hydrogenoformans CooCYP_360059 Carboxydothermus hydrogenoformans

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc. Natl. Acad. Sci. USA 103:17921-17926 (2006)). Thisorganism contains two sets of genes that encode ACS/CODH activity(Rother and Metcalf, Proc. Natl. Acad. Sci. USA 101:16929-16934 (2004)).The protein sequences of both sets of M. acetivorans genes can beidentified by the following GenBank accession numbers.

Protein GenBank ID Organism AcsC NP_618736 Methanosarcina acetivoransAcsD NP_618735 Methanosarcina acetivorans AcsF, CooC NP_618734Methanosarcina acetivorans AcsB NP_618733 Methanosarcina acetivoransAcsEps NP_618732 Methanosarcina acetivorans AcsA NP_618731Methanosarcina acetivorans AcsC NP_615961 Methanosarcina acetivoransAcsD NP_615962 Methanosarcina acetivorans AcsF, CooC NP_615963Methanosarcina acetivorans AcsB NP_615964 Methanosarcina acetivoransAcsEps NP_615965 Methanosarcina acetivorans AcsA NP_615966Methanosarcina acetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (thatis, K_(m), V_(max), k_(cat)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol. 188:463-472(2007)).

In both M. thermoacetica and C. hydrogenoformans, additional CODHencoding genes are located outside of the ACS/CODH operons. Theseenzymes provide a means for extracting electrons (or reducingequivalents) from the conversion of carbon monoxide to carbon dioxide.The reducing equivalents are then passed to acceptors such as oxidizedferredoxin, NADP+, water, or hydrogen peroxide to form reducedferredoxin, NADPH, H₂, or water, respectively. In some cases,hydrogenase encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that is proposed to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO to CO₂ and H₂ (Fox et al., J. Bacteriol. 178:6200-6208(1996)). The CODH-I of C. hydrogenoformans and its adjacent genes havebeen proposed to catalyze a similar functional role based on theirsimilarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al.,PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shownto exhibit intense CO oxidation and CO₂ reduction activities when linkedto an electrode (Parkin et al., J. Am. Chem. Soc. 129:10328-10329(2007)). The genes encoding the C. hydrogenoformans CODH-II and CooF, aneighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMSMicrobiol. Lett. 191:243-247 (2000)). The resulting complex wasmembrane-bound, although cytoplasmic fractions of CODH-II were shown tocatalyze the formation of NADPH suggesting an anabolic role(Svetlitchnyi et al., J. Bacteriol. 183:5134-5144 (2001)). The crystalstructure of the CODH-II is also available (Dobbek et al., Science293:1281-1285 (2001)). The protein sequences of exemplary CODH andhydrogenase genes can be identified by the following GenBank accessionnumbers.

Protein GenBank ID Organism CODH (putative) YP_430813 Moorellathermoacetica CODH-I (CooS-I) YP_360644 Carboxydothermushydrogenoformans CooF YP_360645 Carboxydothermus hydrogenoformans HypAYP_360646 Carboxydothermus hydrogenoformans CooH YP_360647Carboxydothermus hydrogenoformans CooU YP_360648 Carboxydothermushydrogenoformans CooX YP_360649 Carboxydothermus hydrogenoformans CooLYP_360650 Carboxydothermus hydrogenoformans CooK YP_360651Carboxydothermus hydrogenoformans CooM YP_360652 Carboxydothermushydrogenoformans CooM AAC45116 Rhodospirillum rubrum CooK AAC45117Rhodospirillum rubrum CooL AAC45118 Rhodospirillum rubrum CooX AAC45119Rhodospirillum rubrum CooU AAC45120 Rhodospirillum rubrum CooH AAC45121Rhodospirillum rubrum CooF AAC45122 Rhodospirillum rubrum CODH (CooS)AAC45123 Rhodospirillum rubrum CooC AAC45124 Rhodospirillum rubrum CooTAAC45125 Rhodospirillum rubrum CooJ AAC45126 Rhodospirillum rubrumCODH-II YP_358957 Carboxydothermus hydrogenoformans (CooS-II) CooFYP_358958 Carboxydothermus hydrogenoformans

Pyruvate ferredoxin oxidoreductase (PFOR). Anaerobic growth on synthesisgas and methanol in the absence of an external electron acceptor isconferred upon the host organism with MTR and ACS/CODH activity byallowing pyruvate synthesis via pyruvate ferredoxin oxidoreductase(PFOR). The PFOR from Desulfovibrio africanus has been cloned andexpressed in E. coli, resulting in an active recombinant enzyme that wasstable for several days in the presence of oxygen (Pieulle et al., J.Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relativelyuncommon in PFORs and is believed to be conferred by a 60 residueextension in the polypeptide chain of the D. africanus enzyme. The M.thermoacetica PFOR is also well characterized (Menon and Ragsdale,Biochemistry 36:8484-8494 (1997)) and was shown to have high activity inthe direction of pyruvate synthesis during autotrophic growth (Furduiand Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)). Further, E. colipossesses an uncharacterized open reading frame, ydbK, that encodes aprotein that is 51% identical to the M. thermoacetica PFOR. Evidence forpyruvate oxidoreductase activity in E. coli has been described(Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)). The proteinsequences of these exemplary PFOR enzymes can be identified by thefollowing GenBank accession numbers. Several additional PFOR enzymeshave been described (Ragsdale, Chem. Rev. 103:2333-2346 (2003)).

Protein GenBank ID Organism Por CAA70873.1 Desulfovibrio africanus PorYP_428946.1 Moorella thermoacetica YdbK NP_415896.1 Escherichia coli

Hydrogenase (Hyd). Unlike the redox neutral conversion of CO andmethanol to acetyl-CoA or acetate, the production of more highly reducedproducts such as ethanol, butanol, isobutanol, isopropanol,1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, methacrylicacid, adipic acid, and acrylic acid at the highest possible yieldrequires the extraction of additional reducing equivalents from both COand H₂ (for example, see ethanol formation in FIG. 7). Specifically,reducing equivalents (for example, 2[H] in FIG. 6) are obtained by theconversion of CO and water to CO₂ via carbon monoxide dehydrogenase asdescribed in Example II or directly from the activity of ahydrogen-utilizing hydrogenase which transfers electrons from H₂ to anacceptor such as ferredoxin, flavodoxin, FAD+, NAD+, or NADP+.

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J. Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J. Biochem. 156:265-275 (1986); Sawers et al., J. Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities, it ispossible that E. coli or another host organism can provide sufficienthydrogenase activity to split incoming molecular hydrogen and reduce thecorresponding acceptor. Among the endogenous hydrogen-lyase enzymes ofE. coli are hydrogenase 3, a membrane-bound enzyme complex usingferredoxin as an acceptor, and hydrogenase 4, which also uses aferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyfgene clusters, respectively. Hydrogenase activity in E. coli is alsodependent upon the expression of the hyp genes whose correspondingproteins are involved in the assembly of the hydrogenase complexes(Jacobi et al., Arch. Microbiol. 158:444-451 (1992); Rangarajan et al.,J. Bacteriol. 190:1447-1458 (2008)). The M. thermoacetica hydrogenasesare suitable candidates should the production host lack sufficientendogenous hydrogenase activity. M. thermoacetica can grow with CO₂ asthe exclusive carbon source, indicating that reducing equivalents areextracted from H₂ to allow acetyl-CoA synthesis via the Wood-Ljungdahlpathway (Drake, J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res.Microbiol. 155:869-883 (2004); Kellum and Drake, J. Bacteriol.160:466-469 (1984)) (see FIG. 6). M. thermoacetica has homologs toseveral hyp, hyc, and hyf genes from E. coli. These protein sequencesencoded for by these genes can be identified by the following GenBankaccession numbers. In addition, several gene clusters encodinghydrogenase and/or heterodisulfide reductase functionality are presentin M. thermoacetica and their corresponding protein sequences are alsoprovided below.

Hyp assembly proteins.

Protein GenBank ID Organism HypA NP_417206 Escherichia coli HypBNP_417207 Escherichia coli HypC NP_417208 Escherichia coli HypDNP_417209 Escherichia coli HypE NP_417210 Escherichia coli HypFNP_417192 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes.

Protein GenBank ID Organism Moth_2175 YP_431007 Moorella thermoaceticaMoth_2176 YP_431008 Moorella thermoacetica Moth_2177 YP_431009 Moorellathermoacetica Moth_2178 YP_431010 Moorella thermoacetica Moth_2179YP_431011 Moorella thermoacetica Moth_2180 YP_431012 Moorellathermoacetica Moth_2181 YP_431013 Moorella thermoacetica

Hydrogenase 3.

Protein GenBank ID Organism HycA NP_417205 Escherichia coli HycBNP_417204 Escherichia coli HycC NP_417203 Escherichia coli HycDNP_417202 Escherichia coli HycE NP_417201 Escherichia coli HycFNP_417200 Escherichia coli HycG NP_417199 Escherichia coli HycHNP_417198 Escherichia coli HycI NP_417197 Escherichia coli

Hydrogenase 4.

Protein GenBank ID Organism HyfA NP_416976 Escherichia coli HyfBNP_416977 Escherichia coli HyfC NP_416978 Escherichia coli HyfDNP_416979 Escherichia coli HyfE NP_416980 Escherichia coli HyfFNP_416981 Escherichia coli HyfG NP_416982 Escherichia coli HyfHNP_416983 Escherichia coli HyfI NP_416984 Escherichia coli HyfJNP_416985 Escherichia coli HyfR NP_416986 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyc and/or hyf genes.

Protein GenBank ID Organism Moth_2182 YP_431014 Moorella thermoaceticaMoth_2183 YP_431015 Moorella thermoacetica Moth_2184 YP_431016 Moorellathermoacetica Moth_2185 YP_431017 Moorella thermoacetica Moth_2186YP_431018 Moorella thermoacetica Moth_2187 YP_431019 Moorellathermoacetica Moth_2188 YP_431020 Moorella thermoacetica Moth_2189YP_431021 Moorella thermoacetica Moth_2190 YP_431022 Moorellathermoacetica Moth_2191 YP_431023 Moorella thermoacetica Moth_2192YP_431024 Moorella thermoacetica

Additional hydrogenase-encoding gene clusters in M. thermoacetica.

Protein GenBank ID Organism Moth_0439 YP_429313 Moorella thermoaceticaMoth_0440 YP_429314 Moorella thermoacetica Moth_0441 YP_429315 Moorellathermoacetica Moth_0442 YP_429316 Moorella thermoacetica Moth_0809YP_429670 Moorella thermoacetica Moth_0810 YP_429671 Moorellathermoacetica Moth_0811 YP_429672 Moorella thermoacetica Moth_0812YP_429673 Moorella thermoacetica Moth_0813 (possible psuedogene,Moorella thermoacetica GenBank ID unavailable) Moth_0814 YP_429674Moorella thermoacetica Moth_0815 YP_429675 Moorella thermoaceticaMoth_0816 YP_429676 Moorella thermoacetica Moth_1193 YP_430050 Moorellathermoacetica Moth_1194 YP_430051 Moorella thermoacetica Moth_1195YP_430052 Moorella thermoacetica Moth_1196 YP_430053 Moorellathermoacetica Moth_1717 YP_430562 Moorella thermoacetica Moth_1718YP_430563 Moorella thermoacetica Moth_1719 YP_430564 Moorellathermoacetica Moth_1883 YP_430726 Moorella thermoacetica Moth_1884YP_430727 Moorella thermoacetica Moth_1885 YP_430728 Moorellathermoacetica Moth_1886 YP_430729 Moorella thermoacetica Moth_1887YP_430730 Moorella thermoacetica Moth_1888 YP_430731 Moorellathermoacetica Moth_1452 YP_430305 Moorella thermoacetica Moth_1453YP_430306 Moorella thermoacetica Moth_1454 YP_430307 Moorellathermoacetica

This example describes exemplary gene sets for engineering an organismto produce acetyl-CoA from syngas and methanol.

Example XII Cloning, Expression and Activity Assays for Genes andEncoded Enzymes for Engineering an Organism to Produce Acetyl-CoA fromSynthesis Gas and Methanol

This example describes the cloning and expression of genes encodingenzymes that provide a syngas and methanol utilizing organism.

Methanol-methyltransferase (MTR). At least the minimal set of genes, forexample, MtaA, MtaB, and MtaC, for producing Me-THF from methanol arecloned and expressed in E. coli. These genes are cloned viaproof-reading PCR and linked together for expression in a high-copynumber vector such as pZE22-S under control of the repressible PA1-lacO1promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)).Coenzyme B12 is added to the growth medium as these methyltransferaseactivities require cobalamin as a cofactor. Cloned genes are verified byPCR and/or restriction enzyme mapping to demonstrate construction andinsertion of the 3-gene set into the expression vector. DNA sequencingof the presumptive clones is carried out to confirm the expectedsequences of each gene. Once confirmed, the final construct is expressedin E. coli K-12 (MG1655) cells by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) inducer between 0.05 and 1 mM finalconcentration. Expression of the cloned genes is monitored usingSDS-PAGE of whole cell extracts. To determine if expression of theMtaABC proteins confers upon E. coli the ability to transfer methylgroups from methanol to tetrahydrofolate (THF), methanol is fed to therecombinant strain at varying concentrations and its uptake is monitoredalong with methyl-THF synthesis. Activity of the methyltransferasesystem is assayed anaerobically as described for vanillate as a methylsource in M. thermoacetica (Naidu and Ragsdale, J. Bacteriol.183:3276-3281 (2001)) or for the Methanosarcina barkeri methanolmethyltransferase (Sauer et al., Eur. J. Biochem. 243:670-677 (1997);Tallant and Krzycki. J. Bacteriol. 178:1295-1301 (1996); Tallant andKrzycki. J. Bacteriol. 179:6902-6911 (1997); Tallant et al., J. Biol.Chem. 276:4485-4493 (2001)). For a positive control, E. coli cells arecultured in parallel, and endogenous methyltransferase activity ismonitored. Demonstration that activity depends on exogenously addedcoenzyme B12 confirms expression of methanol:corrinoid methyltransferaseactivity in E. coli.

Acetyl-CoA synthase/Carbon monoxide dehydrogenase (ACS/CODH). Usingstandard PCR methods, the entire operons encoding the genes essentialfor ACS/CODH activity from M. thermoacetica, C. hydrogenoformans, and M.acetivorans are assembled into a low or medium copy number vector suchas pZA33-S (P15A-based) or pZS13-S (pSC101-based). As described for themethyltransferase genes, the structure and sequence of the cloned genesare confirmed. Expression is monitored via protein gel electrophoresisof whole-cell lysates grown under strictly anaerobic conditions with therequisite metals (Ni, Zn, Fe) and coenzyme B12 provided. As necessary,the gene cluster is modified for E. coli expression by identificationand removal of any apparent terminators and introduction of consensusribosomal binding sites chosen from sites known to be effective in E.coli (Barrick et al., Nucleic Acids Res. 22:1287-1295 (1994); Ringquistet al., Mol. Microbiol. 6:1219-1229 (1992)). However, each gene clusteris cloned and expressed in a manner parallel to its native structure andexpression. This helps ensure the desired stoichiometry between thevarious gene products, most of which interact with each other. Oncesatisfactory expression of the CODH/ACS gene cluster under anaerobicconditions is achieved, the ability of cells expressing these genes tofix CO and/or CO₂ into cellular carbon is assayed. Initial conditionsemploy strictly anaerobically grown cells provided with exogenousglucose as a carbon and energy source via substrate-levelphosphorylation or anaerobic respiration with nitrate as an electronacceptor. Additionally, exogenously provided CH₃-THF is added to themedium.

Assaying activity of the combined MTR and ACS/CODH pathway. The ACS/CODHgenes as described in Example II are cloned and expressed in cells alsoexpressing the methanol-methyltransferase system also as described inExample II. This is achieved by introduction of compatible plasmidsexpressing ACS/CODH into MTR-expressing cells. For added long-termstability, the ACS/CODH and MTR genes can also be integrated into thechromosome. After strains of E. coli capable of utilizing methanol toproduce Me-THF and of expressing active CODH/ACS gene are made, they areassayed for the ability to utilize both methanol and syngas forincorporation into cell mass and acetate. Initial conditions employstrictly anaerobically grown cells provided with exogenous glucose as acarbon and energy source. Alternatively, or in addition to glucose,nitrate can be added to the fermentation broth to serve as an electronacceptor and initiator of growth. Anaerobic growth of E. coli on fattyacids, which are ultimately metabolized to acetyl-CoA, has beendemonstrated in the presence of nitrate (Campbell et al., Mol.Microbiol. 47:793-805 (2003)). Similar conditions can be employed byculturing the microbial organisms in the presence of an electronacceptor such as nitrate. Oxygen can also be provided as long as itsintracellular levels are maintained below any inhibition threshold ofthe engineered enzymes. “Synthetic syngas” of a composition suitable forthese experiments is employed along with methanol. ¹³C-labeled methanolor ¹³C-labeled CO are provided to the cells, and analytical massspectrometry is employed to measure incorporation of the labeled carboninto acetate and cell mass, for example, proteinogenic amino acids.

Pyruvate ferredoxin oxidoreductase. The pyruvate ferredoxinoxidoreductase genes from M. thermoacetica, D. africanus, and E. coliare cloned and expressed in strains exhibiting MTR and ACS/CODHactivities. Conditions, promoters, and the like, are described above.Given the large size of the PFOR genes and oxygen sensitivity of thecorresponding enzymes, tests are performed using low or single-copyplasmid vectors or single-copy chromosomal integrations. Activity assays(as described in Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499(2000)) are applied to demonstrate activity. In addition, demonstrationof growth on the gaseous carbon sources and methanol in the absence ofan external electron acceptor provides further evidence for PFORactivity in vivo.

Hydrogenase. The endogenous hydrogen-utilizing hydrogenase activity ofthe host organism is tested by growing the cells as described above inthe presence and absence of hydrogen. If a dramatic shift towards theformation of more reduced products during fermentation is observed (forexample, increased ethanol as opposed to acetate), this indicates thatendogenous hydrogenase activity is sufficiently active. In this case, noheterologous hydrogenases are cloned and expressed. If the nativeenzymes do not have sufficient activity or reduce the needed acceptor,the genes encoding an individual hydrogenase complex are cloned andexpressed in strains exhibiting MTR, ACS/CODH, and PFOR activities.Conditions, promoters, and the like, are described above.

This example describes the cloning and expression of genes conferring asyngas and methanol utilization pathway and assay for appropriateactivities.

Example XIII Development and Optimization of Fermentation Process forProduction of Acetyl-CoA from an Organism Engineered to Utilize Syngasand Methanol

This example describes development and optimization of fermentationconditions for syngas and methanol utilizing organisms.

Important process considerations for a syngas fermentation are highbiomass concentration and good gas-liquid mass transfer (Bredwell etal., Biotechnol. Prog. 15:834-844 (1999)). The solubility of CO in wateris somewhat less than that of oxygen. Continuously gas-spargedfermentations can be performed in controlled fermenters with constantoff-gas analysis by mass spectrometry and periodic liquid sampling andanalysis by GC and HPLC. The liquid phase can function in batch mode.Fermentation products such as alcohols, organic acids, and residualglucose along with residual methanol are quantified by HPLC (Shimadzu,Columbia Md.), for example, using an Aminex® series of HPLC columns (forexample, HPX-87 series) (BioRad, Hercules Calif.), using a refractiveindex detector for glucose and alcohols, and a UV detector for organicacids. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm). All piping in these systems is glass ormetal to maintain anaerobic conditions. The gas sparging is performedwith glass frits to decrease bubble size and improve mass transfer.Various sparging rates are tested, ranging from about 0.1 to 1 vvm(vapor volumes per minute). To obtain accurate measurements of gasuptake rates, periodic challenges are performed in which the gas flow istemporarily stopped, and the gas phase composition is monitored as afunction of time.

In order to achieve the overall target productivity, methods of cellretention or recycle are employed. One method to increase the microbialconcentration is to recycle cells via a tangential flow membrane from asidestream. Repeated batch culture can also be used, as previouslydescribed for production of acetate by Moorella (Sakai et al., J.Biosci. Bioeng. 99:252-258 (2005)). Various other methods can also beused (Bredwell et al., Biotechnol. Prog. 15:834-844 (1999); Datar etal., Biotechnol. Bioeng. 86:587-594 (2004)). Additional optimization canbe tested such as overpressure at 1.5 atm to improve mass transfer(Najafpour and Younesi, Enzyme and Microbial Technology 38:223-228(2006)).

Once satisfactory performance is achieved using pure H₂/CO as the feed,synthetic gas mixtures are generated containing inhibitors likely to bepresent in commercial syngas. For example, a typical impurity profile is4.5% CH₄, 0.1% C₂H₂, 0.35% C₂H₆, 1.4% C₂H₄, and 150 ppm nitric oxide(Datar et al., Biotechnol. Bioeng. 86:587-594 (2004)). Tars, representedby compounds such as benzene, toluene, ethylbenzene, p-xylene, o-xylene,and naphthalene, are added at ppm levels to test for any effect onproduction. For example, it has been shown that 40 ppm NO is inhibitoryto C. carboxidivorans (Ahmed and Lewis, Biotechnol. Bioeng. 97:1080-1086(2007)). Cultures are tested in shake-flask cultures before moving to afermentor. Also, different levels of these potential inhibitorycompounds are tested to quantify the effect they have on cell growth.This knowledge is used to develop specifications for syngas purity,which is utilized for scale up studies and production. If any particularcomponent is found to be difficult to decrease or remove from syngasused for scale up, an adaptive evolution procedure is utilized to adaptcells to tolerate one or more impurities.

This example describes development and optimization of fermentationconditions for syngas and methanol utilizing organisms.

Example XIV Methods for Handling CO and Anaerobic Cultures

This example describes methods for handling CO and anaerobic cultures.

Handling of CO in small quantities for assays and small cultures. CO isan odorless, colorless and tasteless gas that is a poison. Therefore,cultures and assays that utilize CO can require special handling.Several assays, including CO oxidation, acetyl-CoA synthesis, COconcentration using myoglobin, and CO tolerance/utilization in smallbatch cultures, call for small quantities of the CO gas that can bedispensed and handled within a fume hood. The biochemical assays calledfor saturating very small quantities (<2 ml) of the biochemical assaymedium or buffer with CO and then performing the assay. All of the COhandling steps were performed in a fume hood with the sash set at theproper height and blower turned on; CO was dispensed from a compressedgas cylinder and the regulator connected to a Schlenk line. The latterensures that equal concentrations of CO will be dispensed to each ofseveral possible cuvettes or vials. The Schlenk line was set upcontaining an oxygen scrubber on the input side and an oil pressurerelease bubbler and vent on the other side. Alternatively, a cold trapcan be used. Assay cuvettes were both anaerobic and CO-containing.Therefore, the assay cuvettes were tightly sealed with a rubber stopperand reagents added or removed using gas-tight needles and syringes.Secondly, small (˜50 ml) cultures were grown with saturating CO intightly stoppered serum bottles. As with the biochemical assays, theCO-saturated microbial cultures were equilibrated in the fume hood usingthe Schlenk line setup. Both the biochemical assays and microbialcultures were in portable, sealed containers and in small volumes makingfor safe handling outside of the fume hood. The compressed CO tank wasadjacent to the fume hood.

Typically, a Schlenk line was used to dispense CO to cuvettes, eachvented. Rubber stoppers on the cuvettes are pierced with 19 or 20 gagedisposable syringe needles and are vented with the same. An oil bubbleris used with a CO tank and oxygen scrubber. The glass or quartzspectrophotometer cuvettes have a circular hole on top into which aKontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unitwas positioned proximal to the fume hood.

Handling of CO in larger quantities fed to large-scale cultures.Fermentation cultures are fed either CO or a mixture of CO and H₂ tosimulate syngas or syngas as a feedstock in fermentative production.Therefore, quantities of cells ranging from 1 liter to several literscan include the addition of CO gas to increase the dissolvedconcentration of CO in the medium. In these circumstances, fairly largeand continuously administered quantities of CO gas will be added to thecultures. At different points, the cultures are harvested or samplesremoved. Alternatively, cells can be harvested with an integratedcontinuous flow centrifuge that is part of the fermenter.

The fermentative processes are generally carried out under anaerobicconditions. In some cases, it is uneconomical to pump oxygen or air intofermenters to ensure adequate oxygen saturation to provide a respiratoryenvironment. In addition, the reducing power generated during anaerobicfermentation is likely to be needed in product formation rather thanrespiration. Furthermore, many of the enzymes being considered forvarious pathways are oxygen-sensitive to varying degrees. Classicacetogens such as M. thermoacetica are obligate anaerobes and theenzymes in the Wood-Ljungdahl pathway are highly sensitive toirreversible inactivation by molecular oxygen. While there areoxygen-tolerant acetogens, the repertoire of enzymes in theWood-Ljungdahl pathway are likely to all have issues in the presence ofoxygen because most are metallo-enzymes, key components are ferredoxins,and regulation may divert metabolism away from the Wood-Ljungdahlpathway to maximize energy acquisition. At the same time, cells inculture act as oxygen scavengers that moderate the need for extrememeasures in the presence of large cell growth.

Anaerobic chamber and conditions. Exemplary anaerobic chambers areavailable commercially (see, for example, Vacuum Atmospheres Company,Hawthorne Calif.; MBraun, Newburyport Mass.). Exemplary conditionsinclude an O₂ concentration of 1 ppm or less and 1 atm pure N₂. In oneexample, 3 oxygen scrubbers/catalyst regenerators can be used, and thechamber can include an O₂ electrode (such as Teledyne; City of IndustryCalif.). Nearly all items and reagents are cycled 4× in the airlock ofthe chamber prior to opening the inner chamber door. Reagents with avolume >5 ml are sparged with pure N₂ prior to introduction into thechamber. Gloves are changed ˜2×/yr and the catalyst containers areregenerated periodically when the chamber displays increasingly sluggishresponse to changes in oxygen levels. The chamber's pressure iscontrolled through one-way valves activated by solenoids. This featureis very convenient because it allows setting the chamber pressure at alevel higher than the surroundings to allow transfer of very small tubesthrough the purge valve.

The anaerobic chambers can achieve levels of O₂ that can be reached thatare consistently very low and are needed for highly oxygen sensitiveanaerobic conditions. However, growth and handling of cells does notusually require such precautions. In an alternative anaerobic chamberconfiguration, platinum or palladium can be used as a catalyst thatrequires some hydrogen gas in the mix. Instead of using solenoid valves,pressure release is controlled by a bubbler. Instead of usinginstrument-based O2 monitoring, test strips can be used instead. Toimprove the anaerobic conditions a few relatively simple changes in oursystem can be made; some are already in progress.

Anaerobic microbiology. Small cultures are handled as described abovefor CO handling. In particular, serum or media bottles are fitted withthick rubber stoppers and aluminum crimps are employed to seal thebottle. Medium, such as Terrific Broth, is made in a conventional mannerand dispensed to an appropriately sized serum bottle. The bottles aresparged with nitrogen for ˜30 min of moderate bubbling. This removesmost of the oxygen from the medium and, after this step; each bottle iscapped with a rubber stopper (such as Bellco 20 mm septum stoppers;Bellco, Vineland, N.J.) and crimp-sealed (Bellco 20 mm). Then thebottles of medium are autoclaved using a slow (liquid) exhaust cycle. Atleast sometimes a needle can be poked through the stopper to provideexhaust during autoclaving; the needle needs to be removed immediatelyupon removal from the autoclave. The sterile medium has the remainingmedium components, for example buffer or antibiotics, added via syringeand needle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl can be added. This was made by weighing the sodium sulfideinto a dry beaker and the cysteine into a serum bottle, bringing bothinto the anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleshould be stoppered immediately as the sodium sulfide solution willgenerate hydrogen sulfide gas upon contact with the cysteine. Wheninjecting into the culture, a syringe filter is used to sterilize thesolution. Other components can be added through syringe needles, such asB12 (10 μM cyanocobalamin), nickel chloride (NiCl₂, 20 microM finalconcentration from a 40 mM stock made in anaerobic water in the chamberand sterilized by autoclaving or by using a syringe filter uponinjection into the culture), and ferrous ammonium sulfate (finalconcentration needed is 100 μM—made as 100-1000× stock solution inanaerobic water in the chamber and sterilized by autoclaving or by usinga syringe filter upon injection into the culture). To facilitate fastergrowth under anaerobic conditions, the 1 l bottles were inoculated with50 ml of a preculture grown anaerobically. Induction of the pA1-lacO1promoter in the vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for ˜3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles can be incubated inan incubator in room air but with continuous nitrogen sparging into thebottles.

This example describes the handling of CO and anaerobic cultures.

Example XV CO Oxidation (CODH) Assay

This example describes assay methods for measuring CO oxidation (COdehydrogenase; CODH).

The 7 gene CODH/ACS operon of Moorella thermoacetica was cloned into E.coli expression vectors. The intact ˜10 kbp DNA fragment was cloned, andit is likely that some of the genes in this region are expressed fromtheir own endogenous promoters and all contain endogenous ribosomalbinding sites. M. thermoacetica is Gram positive, and ribosome bindingsite elements are expected to work well in E. coli. These clones wereassayed for CO oxidation, using an assay that quantitatively measuresCODH activity. Antisera to the M. thermoacetica gene products was usedfor Western blots to estimate specific activity. This activity,described below in more detail, was estimated to be ˜ 1/50th of the M.thermoacetica specific activity.

It is possible that CODH activity of recombinant E. coli cells could belimited by the fact that M. thermoacetica enzymes have temperatureoptima around 55° C. Therefore, a mesophilic CODH/ACS pathway could beadvantageous such as the close relative of Moorella that is mesophilicand does have an apparently intact CODH/ACS operon and a Wood-Ljungdahlpathway, Desulfitobacterium hafniense. Acetogens as potential hostorganisms include, but are not limited to, Rhodospirillum rubrum,Moorella thermoacetica and Desulfitobacterium hafniense.

CO oxidation is both the most sensitive and most robust of the CODH/ACSassays. It is likely that an E. coli-based syngas using system willultimately need to be about as anaerobic as Clostridial (i.e., Moorella)systems, especially for maximal activity. Improvement in CODH should bepossible but will ultimately be limited by the solubility of CO gas inwater.

Initially, each of the genes was cloned individually into expressionvectors. Combined expression units for multiple subunits/1 complex weregenerated. Expression in E. coli at the protein level was determined.Both combined M. thermoacetica CODH/ACS operons and individualexpression clones were made.

CO oxidation assay. This assay is one of the simpler, reliable, and moreversatile assays of enzymatic activities within the Wood-Ljungdahlpathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955(2004)). A typical activity of M. thermoacetica CODH specific activityis 500 U at 55° C. or ˜60 U at 25° C. This assay employs reduction ofmethyl viologen in the presence of CO. This is measured at 578 nm instoppered, anaerobic, glass cuvettes.

In more detail, glass rubber stoppered cuvettes were prepared afterfirst washing the cuvette 4× in deionized water and 1× with acetone. Asmall amount of vacuum grease was smeared on the top of the rubbergasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga.needle plus an exhaust needle. A volume of 0.98 ml of reaction buffer(50 mM Hepes, pH 8.5, 2 mM dithiothreitol (DTT) was added using a 22 Ga.needle, with exhaust needled, and 100% CO. Methyl viologen (CH₃viologen) stock was 1 M in water. Each assay used 20 microliters for 20mM final concentration. When methyl viologen was added, an 18 Ga needle(partial) was used as a jacket to facilitate use of a Hamilton syringeto withdraw the CH₃ viologen. 4-5 aliquots were drawn up and discardedto wash and gas equilibrate the syringe. A small amount of sodiumdithionite (0.1 M stock) was added when making up the CH₃ viologen stockto slightly reduce the CH₃ viologen. The temperature was equilibrated to55° C. in a heated Olis spectrophotometer (Bogart Ga.). A blank reaction(CH₃ viologen+buffer) was run first to measure the base rate of CH₃viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91(CODH-ACS operon of M. thermoacetica with and without, respectively, thefirst cooC). 10 microliters of extract were added at a time, mixed andassayed. Reduced CH₃ viologen turns purple. The results of an assay areshown in Table X

TABLE 2 Crude extract CO Oxidation Activities. ACS90 7.7 mg/ml ACS9111.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2 mg/ml Extract Vol OD/ U/ml U/mgACS90 10 microliters 0.073 0.376 0.049 ACS91 10 microliters 0.096 0.4940.042 Mta99 10 microliters 0.0031 0.016 0.0014 ACS90 10 microliters0.099 0.51 0.066 Mta99 25 microliters 0.012 0.025 0.0022 ACS91 25microliters 0.215 0.443 0.037 Mta98 25 microliters 0.019 0.039 0.004ACS91 10 microliters 0.129 0.66 0.056 Averages ACS90 0.057 U/mg ACS910.045 U/mg Mta99 0.0018 U/mg 

Mta98/Mta99 are E. coli MG1655 strains that express methanolmethyltransferase genes from M. thermoacetia and, therefore, arenegative controls for the ACS90 ACS91 E. coli strains that contain M.thermoacetica CODH operons.

If ˜1% of the cellular protein is CODH, then these figures would beapproximately 100× less than the 500 U/mg activity of pure M.thermoacetica CODH. Actual estimates based on Western blots are 0.5% ofthe cellular protein, so the activity is about 50× less than for M.thermoacetica CODH. Nevertheless, this experiment did clearlydemonstrate CO oxidation activity in recombinant E. coli with a muchsmaller amount in the negative controls. The small amount of COoxidation (CH₃ viologen reduction) seen in the negative controlsindicates that E. coli may have a limited ability to reduce CH₃viologen.

To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGEfollowed by Western blot analyses were performed on the same cellextracts used in the CO oxidation, ACS, methyltransferase, and corrinoidFe—S assays. The antisera used were polyclonal to purified M.thermoacetica CODH-ACS and Mtr proteins and were visualized using analkaline phosphatase-linked goat-anti-rabbit secondary antibody. TheWesterns were performed and results are shown in FIGS. 9A and 9B. Theamounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparisonto the control lanes. Expression of CODH-ACS operon genes including 2CODH subunits and the methyltransferase were confirmed via Western blotanalysis. Therefore, the recombinant E. coli cells express multiplecomponents of a 7 gene operon. In addition, both the methyltransferaseand corrinoid iron sulfur protein were active in the same recombinant E.coli cells. These proteins are part of the same operon cloned into thesame cells.

The CO oxidation assays were repeated using extracts of Moorellathermoacetica cells for the positive controls. Though CODH activity inE. coli ACS90 and ACS91 was measurable, it was at about 130-150× lowerthan the M. thermoacetica control. The results of the assay are shown inFIG. 10. Briefly, cells (M. thermoacetica or E. coli with the CODH/ACSoperon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extractsprepared as described above. Assays were performed as described above at55° C. at various times on the day the extracts were prepared. Reductionof methylviologen was followed at 578 nm over a 120 sec time course.

These results describe the CO oxidation (CODH) assay and results.Recombinant E. coli cells expressed CO oxidation activity as measured bythe methyl viologen reduction assay.

Example XVI Acetyl-CoA Synthase (ACS) Activity Assay (CO Exchange Assay)

This example describes an ACS assay method.

This assay measures the ACS-catalyzed exchange of the carbonyl group ofacetyl-CoA with CO (Raybuck et al., Biochemistry 27:7698-7702 (1988)).ACS (as either a purified enzyme or part of a cell extract) is incubatedwith acetyl-CoA labeled with ¹⁴C at the carbonyl carbon under a COatmosphere. In the presence of active ACS, the radioactivity in theliquid phase of the reaction decreases exponentially until it reaches aminimum defined by the equilibrium between the levels of ¹⁴C-labeledacetyl-CoA and ¹⁴C-labeled CO. The same cell extracts of E. coli MG1655expressing ACS90 and ACS91 employed in the other assays as well ascontrol extracts were assayed by this method.

Briefly in more detail, in small assay vials under normal atmosphere, asolution of 0.2 mM acetyl-CoA, 0.1 mM methyl viologen, and 2 mM Ti(III)citrate in 0.3M MES buffer, pH 6.0, was made. The total reaction volumewhen all components are added was 500 μl. Vials were sealed with rubberstoppers (Bellco) and crimp aluminum seals (Bellco) to create agas-tight reaction atmosphere. Each vial was sparged with 100% CO forseveral minutes, long enough to completely exchange the vials'atmosphere, and brought into an anaerobic chamber. The assay vials wereplaced in a 55° C. sand bath and allowed to equilibrate to thattemperature. A total of 10 scintillation vials with 40 μl of 1M HCl wereprepared for each assay vial. A gas-tight Hamilton syringe was used toadd ACS to the assay vial and incubated for approximately 2-3 minutesfor the reaction to come to equilibrium. A gas-tight Hamilton syringewas used to add 1 μl (0.36 nmoles) ¹⁴C-acetyl-CoA to start the assay(time=0 min). Time points were taken starting immediately. Samples (40μl) were removed from the assay vials with a gas-tight Hamilton syringe.Each sample was added to the 40 μl of HCl in the prepared scintillationvials to quench the reaction. As the ACS enzyme transfers ¹⁴C label toCO from acetyl-CoA, the concentration of the isotope decreasesexponentially. Therefore, the assay was sampled frequently in the earlytime points. The precise time for each sample was recorded. The exactpace of the reaction depends on the ACS enzyme, but generally severalsamples are taken immediately and sampled over the initial 10-15minutes. Samples are continued to be taken for 1-2 hours.

In a particular exemplary assay, four assay conditions were used: blank(no ACS), 12 μl of purified E. coli strains expressing M. thermoaceticaACS, 4 μl of purified E. coli ACS, and 3.7 μl of M. thermoaceticaCODH/ACS. In another exemplary assay, four assay conditions were used:108 μg CODH/ACS, 1 mg Mta99 cell extract, 1 mg ACS90 cell extract, and 1mg ACS91 cell extract. The enzymes were added as 100 μl solutions (50 mMKPi, 0.1M NaCl, pH7.6). A more sensitive assay that can be used for mostof the CODH-ACS activities is the synthesis assay described below.

This example describes the assay conditions for measuring ACS activity.

Example XVII Acetyl-CoA Synthesis and Methyltransferase Assays

This example describes acetyl-CoA synthesis and methyltransferaseassays.

Synthesis assay. This assay is an in vitro reaction that synthesizesacetyl-CoA from methyl-tetrahydrofolate, CO, and CoA using CODH/ACS,methyltransferase (MeTr), and corrinoid Fe—S protein (CFeSP) (Raybuck etal., Biochemistry 27:7698-7702 (1988)). By adding or leaving out each ofthe enzymes involved, this assay can be used for a wide range ofexperiments, from testing one or more purified enzymes or cell extractsfor activity, to determining the kinetics of the reaction under variousconditions or with limiting amounts of substrate or enzyme. Samples ofthe reaction taken at various time points are quenched with 1M HCl,which liberates acetate from the acetyl-CoA end product. Afterpurification with Dowex columns, the acetate can be analyzed bychromatography, mass spectrometry, or by measuring radioactivity. Theexact method will be determined by the specific substrates used in thereaction.

A ¹⁴C-labeled methyl-THF was utilized, and the radioactivity of theisolated acetate samples was measured. The primary purpose was to testCFeSP subunits. The assay also included +/−purified methyltransferaseenzymes. The following 6 different conditions were assayed: (1) purifiedCODH/ACS, MeTr, and CFeSP as a positive control; (2) purified CODH/ACSwith ACS90 cell extract; (3) purified CODH/ACS with ACS91 cell extract;(4) purified CODH/ACS, MeTr with ACS90 cell extract; (5) purifiedCODH/ACS, MeTr with ACS91 cell extract; (6) purified CODH/ACS, MeTr withas much ACS91 cell extract as possible (excluding the MES buffer).

The reaction is assembled in the anaerobic chamber in assay vials thatare filled with CO. The total reaction volume is small compared to thevial volume, so the reagents can be added before or after the vial isfilled with CO, so long as a gas-tight Hamilton syringe is used and thereagents are kept anaerobic. The reaction (˜60 ul total) consisted ofthe cell extract (except assay #1), CoA, Ti(III) citrate, MES (exceptassay #6), purified CODH/ACS, ¹⁴C-methyl-tetrahydrofolate,methyl-viologen, and ferredoxin. Additionally, purified MeTr was addedto assays #1 and #4-6, and purified CFeSP was added to assay #1.

The reaction was carried out in an anaerobic chamber in a sand bath at55° C. The final reagent added was the ¹⁴C-methyl-tetrahydrofolate,which started the reaction (t=0s). An initial sample was takenimmediately, followed by samples at 30 minutes, 1 hour, and 2 hours.These time points are not exact, as the 6 conditions were runconcurrently (since this experiment was primarily a qualitative one).The 15 μl samples were added to 15 μl of 1M HCl in scintillation vials.For the last sample, if less than 15 μl was left in the reactions, theassay vials were rinsed with the 15 ul of HCl to take the remainder ofthe reaction. A volume of 10 μl of cell extract was used for assay #2-5,and 26.4 μl of cell extract was used for assay #6.

Typical amounts of purified enzyme to be used in the assays is asfollows: CODH/ACS=˜0.2 nmoles; MeTr=0.2 nmoles; CFeSP=0.05 nmoles.Typical assay concentrations are used as follows: CODH/ACS=1 uM;Me-CFeSP=0.4 uM; MeTr=1 uM; Ferredoxin=3 uM; CoA=0.26 mM; ¹⁴Cmethyl-THF=0.4 mM; methyl viologen=0.1 mM; and Ti(III) citrate=3 mM.

After counting the reaction mixtures, it was determined that thecorrinoid Fe—S protein in ACS90 extracts was active with total activityapproaching approximately ⅕ of the positive control and significantlyabove the negative control (no extract).

A non-radioactive synthesis assay can also be used. Optionalnon-radioactive assay conditions are as follows: Assay condition #1: 100mM MES, pH6.0; 1 mM CoA; 1 mM Me-THF; 0.33 mM Ti(III) citrate, volume to950 ul, +50 ul of extract; incubated under a CO atmosphere (Ar forcontrol), at 55° C. These reactions should be carried out in the dark,as the corrinoid methyl carrier is light sensitive. Assay condition #2:100 mM MES, pH6.0; 1 mM CoA; 1 mM Me-THF; 1 mM methyl viologen; volumeto 950 ul, +50 ul of extract; incubated under a CO atmosphere, at 55°C., in the dark. The reaction was quenched with 10 μl of 10% formicacid, with samples taken at 1 hr, 3 hrs, and 6.5 hrs, and stored at−20°. Assay condition #3: 100 mM Tris, pH 7.6; 5 mM CoA; 7.5 mM Me-THF;1 mM Me-viologen; volume to 90 μl, +10 μl extract; incubated under CO orAr, at 55° C. in the dark for 1 hr, quenched with 10 μl 10% formic acid,and stored at −20° C.

In Lu et al., (J. Biol. Chem. 265:3124-3133. (1990)), the pH optimum forthe synthesis reaction was found to be between 7.2-7.5. Lu et al. alsofound that CoA concentrations above 10 mM were inhibitory. Lu et al.described using methyl iodide as the methyl donor instead of Me-THF, andused 5-7.5 mM concentrations. Lu et al. also determined that DTT orother reducing agents were not necessary, although they did useferredoxin as an electron carrier. Methyl viologen was substituted inthe above-described reactions. In addition, Maynard et al., J. Biol.Inorg. Chem. 9:316-322 (2004), has determined that the electron carrierwas not strictly necessary, but that failure to include one resulted ina time lag of the synthesis. Maynard et al. used 1 mM methyl viologen aselectron carrier when one was used.

Methyltransferase Assay. Within the CODH-ACS operon is encoded anessential methyltransferase activity that catalyzes the transfer of CH₃from methyl-tetrahydrofolate to the ACS complex as part of the synthesisof acetyl-CoA. This is the step that the methyl and carbonyl pathwaysjoin together. Within the operon in M. thermoacetica, the Mtr-encodinggene is Moth_(—)1197 and comes after the main CODH and ACS subunits.Therefore, Mtr activity would constitute indirect evidence that the moreproximal genes can be expressed.

Mtr activity was assayed by spectroscopy. Specifically, methylatedCFeSP, with Co(III), has a small absorption peak at ˜450 nm, whilenon-methylated CFeSP, with Co(I), has a large peak at ˜390 nm. Thisspectrum is due to both the cobalt and iron-sulfur cluster chromophores.Additionally, the CFeSP can spontaneously oxidize to Co(II), whichcreates a broad absorption peak at ˜470 nm (Seravalli et al.,Biochemistry 38:5728-5735 (1999)). Recombinant methyltransferase istested using E. coli cell extracts, purified CFeSP from M.thermoacetica, and methyl-tetrahydrofolate. The methylation of thecorrinoid protein is observed as a decrease in the absorption at 390 nmwith a concurrent increase in the absorption at 450 nm, along with theabsence of a dominant peak at 470 nm.

Non-radioactive assays are also being developed using ¹³C-methanol. Thisshould transfer to tetrahydrofolate and create a MTHF of molecularmass+1. Alternatively, the methyltransferase is thought to also work bytransfer of the methanol methyl group to homocysteine to formmethionine. This assay is also useful because methionine+1 mass is morereadily detectable than MTHF+1 or some other possibilities. In additionto using ¹³C, deuterium can also be used as a tracer, both of which canbe measured using mass spectrometry. These tracers can also be used inin vivo labeling studies. Other assay methods can be used to determinevarious intermediates or products including, for example, electronparamagnetic resonance (EPR), Mossbauer spectroscopy, Electron-NuclearDOuble Resonance (ENDOR), infrared, magnetic circular dichroism (MCD),crystallography, X-ray absorption, as well as kinetic methods, includingstopped flow and freeze-quench EPR.

FIG. 8 illustrates how methanol methyltransferase can be fitted into aCODH/ACS (‘syngas’) pathway. Essentially, the methyl group of methanolis transferred via a cobabalamin-dependent process to tetrahydrofolateand then to the corrinoid-FeS protein of CODH/ACS (also a cobalaminprotein) and that, in turn, donates the methyl group to the ACS reactionthat results in acetate synthesis. The methanol methyltransferasecomplex consists of three gene products; two of these, MtaB and MtaC,(Moth_(—)1209 and Moth_(—)1208) are adjacent and were readily cloned.The third, MtaA, may be encoded by three different genes (Moth_(—)2100,Moth_(—)2102, and Moth_(—)2346), and it unclear whether all three genesare required or whether a subset of the three can function. All cloningin E. coli was performed using the Lutz-Bujard vectors (Lutz and Bujard,Nucleic Acids Res. 25:1203-1210 (1997)).

The following assay can be used to determine the activity of MtaB thatencodes a methanol methyltransferase gene product. A positive controlfor the latter can be performed with vanillate o-demethylation.

Methanol Methyltransferase reaction. An exemplary methanolmethyl-transfer reaction has been described previously (Sauer andThauer, Eur. J. Biochem. 249:280-285 (1997); Naidu and Ragsdale, J.Bacteriol. 183:3276-3281 (2001)). The reaction conditions are asfollows: 50 mM MOPS/KOH, pH 7.0; 10 mM MgCl₂; 4 mM Ti(III) citrate; 0.2%dodecylmaltoside (replacing SDS, see Sauer and. Thauer, Eur. J. Biochem.253:698-705 (1998)); 25 μM hydroxycobalamin; 1% MeOH or 1 mM vanillate(depending on the methyl transferase version).

These reactions are measured by spectrograph readings in the dark at 37°C. or 55° C. This assay tests the ability of MtaB or MtvB to transferthe methyl group to cobalamin from methanol or vanillate, respectively.

Example XVIII E. coli CO Tolerance Experiment and CO Concentration Assay(Myoglobin Assay)

This example describes the tolerance of E. coli for high concentrationsof CO.

To test whether or not E. coli can grow anaerobically in the presence ofsaturating amounts of CO, cultures were set up in 120 ml serum bottleswith 50 ml of Terrific Broth medium (plus reducing solution, NiCl₂,Fe(II)NH₄SO₄, cyanocobalamin, IPTG, and chloramphenicol) as describedabove for anaerobic microbiology in small volumes. One half of thesebottles were equilibrated with nitrogen gas for 30 min. and one half wasequilibrated with CO gas for 30 min. An empty vector (pZA33) was used asa control, and cultures containing the pZA33 empty vector as well asboth ACS90 and ACS91 were tested with both N₂ and CO. All wereinoculated and grown for 36 hrs with shaking (250 rpm) at 37° C. At theend of the 36 hour period, examination of the flasks showed high amountsof growth in all. The bulk of the observed growth occurred overnightwith a long lag.

Given that all cultures appeared to grow well in the presence of CO, thefinal CO concentrations were confirmed. This was performed using anassay of the spectral shift of myoglobin upon exposure to CO. Myoglobinreduced with sodium dithionite has an absorbance peak at 435 nm; thispeak is shifted to 423 nm with CO. Due to the low wavelength and need torecord a whole spectrum from 300 nm on upwards, quartz cuvettes must beused. CO concentration is measured against a standard curve and dependsupon the Henry's Law constant for CO of maximum water solubility=970micromolar at 20° C. and 1 atm.

For the myoglobin test of CO concentration, cuvettes were washed 10×with water, 1× with acetone, and then stoppered as with the CODH assay.N₂ was blown into the cuvettes for ˜10 min. A volume of 1 ml ofanaerobic buffer (HEPES, pH 8.0, 2 mM DTT) was added to the blank (notequilibrated with CO) with a Hamilton syringe. A volume of 10 microlitermyoglobin (˜1 mM—can be varied, just need a fairly large amount) and 1microliter dithionite (20 mM stock) were added. A CO standard curve wasmade using CO saturated buffer added at 1 microliter increments. Peakheight and shift was recorded for each increment. The cultures testedwere pZA33/CO, ACS90/CO, and ACS91/CO. each of these was added in 1microliter increments to the same cuvette. Midway through the experimenta second cuvette was set up and used. The results are shown in Table 3.

TABLE 3 Carbon Monoxide Concentrations, 36 hrs. Strain and GrowthConditions Final CO concentration (micromolar) pZA33-CO 930 ACS90-CO 638494 734 883 ave 687 SD 164 ACS91-CO 728 812 760 611 ave. 728 SD 85

The results shown in Table 3 indicate that the cultures grew whether ornot a strain was cultured in the presence of CO or not. These resultsindicate that E. coli can tolerate exposure to CO under anaerobicconditions and that E. coli cells expressing the CODH-ACS operon canmetabolize some of the CO.

These results demonstrate that E. coli cells, whether expressingCODH/ACS or not, were able to grow in the presence of saturating amountsof CO. Furthermore, these grew equally well as the controls in nitrogenin place of CO. This experiment demonstrated that laboratory strains ofE. coli are insensitive to CO at the levels achievable in a syngasproject performed at normal atmospheric pressure. In addition,preliminary experiments indicated that the recombinant E. coli cellsexpressing CODH/ACS actually consumed some CO, probably by oxidation tocarbon dioxide.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1. A non-naturally occurring microorganism, comprising one or moreexogenous proteins conferring to said microorganism a pathway to convertCO and H₂ or CO₂ and H₂ to acetyl-coenzyme A (acetyl-CoA), wherein saidmicroorganism lacks the ability to convert CO and H₂ to acetyl-CoA inthe absence of said one or more exogenous proteins.
 2. The non-naturallyoccurring microorganism of claim 1, wherein said one or more exogenousproteins is selected from cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase. 3-10.(canceled)
 11. A non-naturally occurring microorganism, comprising oneor more exogenous proteins conferring to said microorganism a pathway toconvert CO and H₂ or CO₂ and H₂ to methyl-tetrahydrofolate (methyl-THF),wherein said microorganism lacks the ability to convert CO and H₂ tomethyl-THF in the absence of said one or more exogenous proteins. 12.The non-naturally occurring microorganism of claim 11, wherein said oneor more exogenous proteins is selected from ferredoxin oxidoreductase,formate dehydrogenase, formyltetrahydrofolate synthetase,methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolatedehydrogenase and methylenetetrahydrofolate reductase.
 13. Thenon-naturally occurring microorganism of claim 12, wherein ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase are each expressed. 14-20. (canceled)
 21. A non-naturallyoccurring microorganism, comprising a genetic modification conferring tosaid microorganism an increased efficiency of producing acetyl-CoA fromCO₂, CO and H₂, or a combination thereof, relative to said microorganismin the absence of said genetic modification, wherein said microorganismcomprises a pathway to convert CO₂, CO and H₂, or a combination thereof,to acetyl-CoA.
 22. The non-naturally occurring microorganism of claim21, wherein said genetic modification comprises expression of one ormore nucleic acid molecules encoding one or more exogenous proteins,whereby expression of said one or more exogenous proteins increases theefficiency of producing acetyl-CoA from CO₂, CO and H₂, or a combinationthereof.
 23. The non-naturally occurring microorganism of claim 22,wherein said one or more exogenous proteins is selected from cobalamidecorrinoid/iron-sulfur protein, methyltransferase, carbon monoxidedehydrogenase, acetyl-coA synthase, acetyl-CoA synthase disulfidereductase and hydrogenase.
 24. The non-naturally occurring microorganismof claim 23, wherein cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-coA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase are eachexpressed.
 25. A non-naturally occurring microbial organism, comprisinga microbial organism having an acetyl-coenzyme A (acetyl-CoA) pathwaycomprising at least one exogenous nucleic acid encoding an acetyl-CoApathway enzyme or protein expressed in a sufficient amount to produceacetyl-CoA, said acetyl-CoA pathway comprisingmethanol-methyltransferase and acetyl-CoA synthase/carbon monoxidedehydrogenase.
 26. The non-naturally occurring microbial organism ofclaim 25, wherein said acetyl-CoA pathway confers the ability to convertCO₂, CO or H₂, or a combination thereof, and methanol to acetyl-CoA. 27.The non-naturally occurring microbial organism of claim 25, wherein themethanol-methyltransferase comprises an enzyme or protein selected frommethanol methyltransferase, corrinoid protein andmethyltetrahydrofolate:corrinoid protein methyltransferase.
 28. Thenon-naturally occurring microbial organism of claim 25, wherein theacetyl-CoA synthase/carbon monoxide dehydrogenase comprises an enzyme orprotein selected from methyltetrahydrofolate:corrinoid proteinmethyltransferase, corrinoid iron-sulfur protein, nickel-proteinassembly protein, ferredoxin, acetyl-CoA synthase, carbon monoxidedehydrogenase and nickel-protein assembly protein.
 29. The non-naturallyoccurring microbial organism of claim 25, wherein themethanol-methyltransferase comprises an enzyme or protein selected frommethanol methyltransferase, corrinoid protein andmethyltetrahydrofolate:corrinoid protein methyltransferase and theacetyl-CoA synthase/carbon monoxide dehydrogenase comprises an enzyme orprotein selected from methyltetrahydrofolate:corrinoid proteinmethyltransferase, corrinoid iron-sulfur protein, nickel-proteinassembly protein, ferredoxin, acetyl-CoA synthase, carbon monoxidedehydrogenase and nickel-protein assembly protein. 30-37. (canceled) 38.The non-naturally occurring microbial organism of claim 25, wherein saidmicrobial organism comprises ten exogenous nucleic acids each encodingan acetyl-CoA pathway enzyme or protein.
 39. The non-naturally occurringmicrobial organism of claim 38, wherein said ten exogenous nucleic acidsencode a methanol-methyltransferase comprising methanolmethyltransferase, corrinoid protein andmethyltetrahydrofolate:corrinoid protein methyltransferase and anacetyl-CoA synthase/carbon monoxide dehydrogenase comprisingmethyltetrahydrofolate:corrinoid protein methyltransferase, corrinoidiron-sulfur protein, nickel-protein assembly protein, ferredoxin,acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-proteinassembly protein.
 40. The non-naturally occurring microbial organism ofclaim 25, wherein said at least one exogenous nucleic acid is aheterologous nucleic acid.
 41. The non-naturally occurring microbialorganism of claim 25, wherein said non-naturally occurring microbialorganism is in a substantially anaerobic culture medium.
 42. Thenon-naturally occurring microbial organism of claim 25, wherein saidnon-naturally occurring microbial organism further comprises pyruvateferredoxin oxidoreductase.
 43. The non-naturally occurring microbialorganism of claim 42, wherein pyruvate ferredoxin oxidoreductase isencoded by an exogenous nucleic acid.
 44. The non-naturally occurringmicrobial organism of claim 25, wherein said non-naturally occurringmicrobial organism further comprises hydrogenase.
 45. The non-naturallyoccurring microbial organism of claim 44, wherein said hydrogenase isencoded by an endogenous nucleic acid.
 46. The non-naturally occurringmicrobial organism of claim 44, wherein said hydrogenase is encoded byan exogenous nucleic acid.
 47. A method for producing acetyl-CoA,comprising culturing the non-naturally occurring microbial organism ofclaim 25 having an acetyl-CoA pathway, said pathway comprising at leastone exogenous nucleic acid encoding an acetyl-CoA pathway enzyme orprotein expressed in a sufficient amount to produce acetyl-CoA, underconditions and for a sufficient period of time to produce acetyl-CoA,said acetyl-CoA pathway comprising methanol-methyltransferase andacetyl-CoA synthase/carbon monoxide dehydrogenase.
 48. The method ofclaim 47, wherein the methanol-methyltransferase comprises an enzyme orprotein selected from methanol methyltransferase, corrinoid protein andmethyltetrahydrofolate:corrinoid protein methyltransferase.
 49. Themethod of claim 47, wherein the acetyl-CoA synthase/carbon monoxidedehydrogenase comprises an enzyme or protein selected frommethyltetrahydrofolate:corrinoid protein methyltransferase, corrinoidiron-sulfur protein, nickel-protein assembly protein, ferredoxin,acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-proteinassembly protein.
 50. The method of claim 47, wherein themethanol-methyltransferase comprises an enzyme or protein selected frommethanol methyltransferase, corrinoid protein andmethyltetrahydrofolate:corrinoid protein methyltransferase and theacetyl-CoA synthase/carbon monoxide dehydrogenase comprises an enzyme orprotein selected from methyltetrahydrofolate:corrinoid proteinmethyltransferase, corrinoid iron-sulfur protein, nickel-proteinassembly protein, ferredoxin, acetyl-CoA synthase, carbon monoxidedehydrogenase and nickel-protein assembly protein.
 51. The method ofclaim 47, wherein said non-naturally occurring microbial organism is ina substantially anaerobic culture medium.
 52. The method of claim 51,wherein said non-naturally occurring microbial organism is cultured inthe presence of CO₂, CO, or H₂, or a combination thereof, and methanol.53-68. (canceled)
 69. The method of claim 51, wherein said non-naturallyoccurring microbial organism is cultured in the presence of nitrate.